ES2743681T3 - Non-human animals expressing antibodies with a common light chain - Google Patents

Abstract

A genetically modified mouse comprising a B lymphocyte expressing a human light chain variable domain (VL) obtained from a rearranged human Vκ1-39 / Jκ sequence that is present in the germ line of the mouse, where the mouse lacks of an endogenous immunoglobulin non-rearranged Vκ gene segment and an endogenous immunoglobulin Jκ gene segment not rearranged; and wherein the human VL domain (or domains) is associated with a human heavy chain variable domain (VH) obtained from a rearranged human VH / DH / JH region selected from 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3-30 / 3- 3/4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6-13 / 4.

Description

DESCRIPTION

Non-human animals expressing antibodies with a common light chain

Area of the invention

A genetically modified mouse is provided that expresses antibodies that have a human variable / mouse constant light chain associated with various human variable / mouse constant heavy chains. A method is provided for making a bispecific antibody from gene sequences of the mouse B-cell variable region.

Background

The antibodies usually comprise a homodimeric component of the heavy chain, wherein each monomer of the heavy chain is associated with an identical one of the light chain. Antibodies that have a heterodimeric heavy chain component (eg, bispecific antibodies) are desirable as therapeutic antibodies. But making bispecific antibodies with a suitable light chain component that can successfully associate with each of the heavy chains of a bispecific antibody has proven problematic.

In one approach, a light chain could be selected by studying the usage statistics for all variable domains of the light chain, identifying the most frequently used chain in human antibodies, and matching said light chain in vitro with the two heavy chains of different specificity

In another approach, a light chain could be selected by observing sequences of the light chain in a phage display library ("phage display library", for example, a phage display library comprising sequences from the variable region of the light chain, by example, a human scFv library), and selecting from the library the variable region of the most commonly used light chain.) The light chain can then be tested on the two different heavy chains of interest.

In another approach, a light chain could be selected by testing a phage display library of variable sequences of the light chain using the variable sequences of the heavy chain of the two heavy chains of interest as probes. A light chain that is associated with both variable sequences of the heavy chain could be selected as a light chain for heavy chains.

In another approach, a candidate light chain could be aligned with the light chains analogous to the heavy chains, and the modifications are made in the light chain to match more closely the characteristics of the sequences common to the light chains analogous to both heavy chains. . If the immunogenicity probabilities need to be minimized, the modifications preferably result in sequences that are present in known sequences of the light chain, such that the proteolytic process is unlikely to generate a T-cell epitope based on parameters and methods. known in the art to assess the likelihood of immunogenicity (i.e. in silico tests in addition to wet).

All of the above approaches are based on in vitro methods that incorporate a number of a priori limitations, for example, sequence identity, the ability to associate with specific preselected heavy chains, etc. There is a need in the art for compositions and methods that are not based on handling in vitro conditions, but instead employ more biologically sensitive approaches to make human epitope-binding proteins that include a common light chain.

Summary

Genetically modified mice are provided that express variable domains of the light and heavy chains of the human immunoglobulin, wherein the mice have a limited repertoire of the limited light chain. In particular, a genetically modified mouse is provided comprising a B lymphocyte that expresses a variable domain of the light chain (Vl) obtained from a rearranged human Vk1-39 / Jk sequence that is present in the mouse germ line , wherein the mouse lacks a non-rearranged Vk gene segment of the endogenous immunoglobulin and a non-rearranged Jk gene segment of the endogenous immunoglobulin; and wherein the domain, or domains, human Vl is associated with a heavy chain variable domain (Vh) obtained from a rearranged human Vh / Dh / Jh region selected from 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6-13 / 4. The use of such a mouse is also envisaged in the manufacture of an antibody comprising a human heavy chain (VH) variable domain obtained from a rearranged human Vh / Dh / Jh region selected from 2-5 / 3. -22/1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1 -1/4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6 -13/4, associated with a variable region of the human light chain obtained from a rearranged human Vk1-39 / Jk sequence. In addition, there is provided a method of producing an antibody for an antigen of interest comprising a variable domain of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region, selected from 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6-13 / 4 associated with a variable region of the human light chain obtained from a rearranged human Vk1-39 / Jk sequence, the method comprising: (a) immunizing the mouse provided with an antigen of interest; (b) obtain from the mouse a variable domain of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region, selected from 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6-13 / 4; and employing the sequence of the variable region of the immunoglobulin obtained in (b) associated with said variable region of the human light chain in an antibody that specifically binds to the antigen of interest.

A genetically modified mouse comprising a B lymphocyte expressing a variable domain of the human light chain (Vl) obtained from a rearranged human Vk3-20 / Jk sequence that is present in the mouse germ line, is also provided. where the mouse lacks a non-rearranged endogenous immunoglobulin Vk gene segment and a non-rearranged endogenous immunoglobulin Jk gene segment; and wherein the domain, or domains, human Vl is associated with a variable domain of the human heavy chain (Vh) obtained from a rearranged human Vh / Dh / Jh region selected from 3-30 / 3-3 / 3 , 3-33 / 1-7 / 4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4. The use of such a mouse is also envisaged in the manufacture of an antibody comprising a human heavy chain (Vh) variable domain obtained from a rearranged human Vh / Dh / Jh region selected from 3-30 / 3-3 / 3, 3-33 / 1-7 / 4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4. The use of such a mouse is also envisaged in the manufacture of an antibody comprising a human heavy chain (Vh) variable domain obtained from a rearranged human Vh / Dh / Jh region selected from 3-30 / 3-3 / 3, 3-33 / 1-7 / 4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4 associated with a variable region of the human light chain obtained from a sequence of Vk3-20 / Jk human rearranged. A method is also provided for producing an antibody to an antigen of interest comprising a human heavy chain variable domain obtained from a rearranged human Vh / Dh / Jh region selected from 3-30 / 3-3 / 3, 3-33 / 1-7 / 4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4 associated with a variable region of the human light chain obtained from a human Vk3-20 / Jk sequence rearranged, the method comprising: (a) immunizing the mouse provided with an antigen of interest; (b) obtain from the mouse a variable domain of the human heavy chain obtained from a rearranged human VH / DH / JH region selected from 3-30 / 3-3 / 3, 3-33 / 1-7 / 4, 3- 33 / 2-15 / 4 or 3-53 / 1-1 / 4; and (c) employing the sequence of the immunoglobulin variable region obtained in (b) associated with said human light chain variable region in an antibody that specifically binds to the antigen of interest.

Reference is also made to a biological system to generate a variable domain of the human light chain that is associated, and expressed, with a diverse repertoire of variable domains of the human heavy chain with affinity maturation. Reference is also made to methods for the elaboration of binding proteins that comprise immunoglobulin variable domains, which comprise immunizing mice that have a limited repertoire of the immunoglobulin light chain with an antigen of interest, and employing a gene sequence of the Variable region of mouse immunoglobulin in a binding protein that specifically binds to the antigen of interest. Methods include methods for making variable domains of the human immunoglobulin heavy chain suitable for use in the production of multispecific antigen binding proteins.

The genetically modified mice provided can select variable domains of the human immunoglobulin heavy chain with suitable affinity maturation, obtained from a repertoire of gene segments of the non-rearranged human heavy chain variable region, where the variable domains of the heavy chain with affinity maturation are associated and expressed with a single variable domain of the human light chain obtained from a gene segment of the variable region of the human light chain. Genetically modified mice are also provided that have a choice of two gene segments of the variable region of the human light chain.

Genetically modified mice are provided that express a limited repertoire of variable domains of the human light chain, or a single variable domain of the human light chain, of a limited repertoire of sequences of the variable region of the human light chain. Mice are engineered to include a single light chain V / J sequence (or two V / J sequences) that express a variable region of a single light chain (or that expresses either or both of two variable regions). The rearranged sequences are a sequence of rearranged human Vk1-39 / Jk, a rearranged human Vk3-20 / Jk, or both. A light chain comprising the variable sequence is capable of pairing with a plurality of human heavy chains with affinity maturation, clonally selected by the mouse, where the variable regions of the heavy chain specifically bind different epitopes.

Reference is also made to a genetically modified mouse comprising a single gene segment of the variable region of the light chain (Vl) of the human immunoglobulin that is capable of being rearranged with a human J gene segment (selected from one or a plurality of segments Jl) and encoding a human Vl domain of an immunoglobulin light chain. In another case, the mouse comprises no more than two Vl gene segments, each of which is capable of rearranging with a human J gene segment (selected from one or a plurality of Jl segments) and encoding a human Vl domain of a light chain of immunoglobulin.

In one case, the only human Vl gene segment is operatively linked to a Jl gene segment. selected from Jk1, Jk2, Jk3, Jk4, and Jk5, where the only human Vl gene segment is able to rearrange to form a sequence that encodes a gene in the light chain variable region with any one or more human Jl gene segments .

In addition, reference is made to a genetically modified mouse comprising an immunoglobulin light chain locus that does not comprise an endogenous mouse Vl gene segment that is capable of being rearranged to form an immunoglobulin light chain gene, wherein the Vl locus comprises a human Vl gene segment that is capable of rearranging to encode a Vl region of a light chain gene. In a specific case, the human Vl gene segment is a human Vk1-39Jk5 gene segment or a human Vk3-20Jk1 gene segment. In addition, reference is made to a genetically modified mouse comprising a Vl locus that does not comprise an endogenous mouse Vl gene segment that is capable of rearranging to form an immunoglobulin light chain gene, wherein the VL locus comprises no more of two human VL gene segments that are capable of rearranging to encode a Vl region of a light chain gene. In a specific case, the no more than 2 human Vl gene segments are a human Vk1-39Jk gene segment and a human Vk3-20Jk gene segment.

In one aspect, the genetically modified mouse provided comprises a single variable region of the light chain (Vl) of the rearranged human immunoglobulin (V / J) (i.e., a Vl / Jl region) encoding a human Vl domain of the chain light of an immunoglobulin. In another aspect, the mouse comprises no more than two rearranged human Vl regions that are capable of encoding a human Vl domain of an immunoglobulin light chain. The rearranged sequences are a sequence of rearranged human Vk1-39 / Jk, a rearranged human Vk3-20 / Jk, or both.

The Vl region is a sequence of human Vk1-39Jk or a sequence of human Vk3-20Jk. In one embodiment, the human Jl segment of the rearranged Vl / Jl sequence is selected from Jk1, Jk2, Jk3, Jk4 and Jk5. In a specific embodiment, the Vl region is a sequence of human Vk1-39Jk5 or a sequence of human Vk3-20Jk1. In a specific embodiment the mouse has both a human Vk1-39Jk5 sequence and a human Vk3-20Jk1 sequence.

In one embodiment, the human Vl gene segment is operatively linked to a mouse or human leader sequence. In one embodiment, the leader sequence is a mouse leader sequence. In a specific embodiment, the mouse leader sequence is a mouse Vk3-7 leader sequence. In a specific embodiment, the leader sequence is operatively linked to the rearranged human Vl / Jl sequence.

In one embodiment, the Vl gene segment is linked to the sequence of an immunoglobulin promoter. In one embodiment, the promoter sequence is a sequence of a human promoter. In a specific embodiment, the human immunoglobulin promoter is a human V k3-15 promoter. In a specific embodiment, the promoter is operatively linked to the rearranged human Vl / Jl sequence.

Reference is also made to a light chain locus comprising a 5 'end of the flanked leader sequence (with respect to the transcription direction of a VL gene segment) with a human immunoglobulin promoter, and a 3' flanked end with a human VL gene segment that is rearranged with a human J segment, and encodes a VL domain of an inverse chimeric light chain comprising a constant region of the endogenous mouse light chain (Cl). In a specific embodiment, the Vl gene segment is located in the mouse Vk locus, and the mouse Cl is a mouse Ck.

In one embodiment, the light chain locus comprises the 5 'end with the flanked leader sequence (with respect to the transcription direction of a VL gene segment) with a human immunoglobulin promoter, a 3' end flanked with a region Human Vl rearranged (sequence Vl / Jl) and encodes a Vl domain of an inverse chimeric light chain comprising a constant region of the endogenous mouse light chain (Cl). In a specific embodiment, the rearranged human Vl / Jl sequence is found in the mouse kappa (k) locus, and the mouse Cl is a mouse Ck.

In one embodiment, the modified mouse locus Vl is a locus of the light chain k, and the locus of the light chain k comprises an intronic enhancer k, a mouse enhancer 3 'k, or both an intronic enhancer and a 3 'enhancer.

In one embodiment, the mouse comprises a lambda light chain locus (á) of the non-functional immunoglobulin. In a specific embodiment, the light chain locus á comprises a deletion of one or more locus sequences, wherein said one or more deletions make the light chain locus á unable to rearrange to form a light chain gene . In another embodiment, all or substantially all of the Vl gene segments of the light chain locus are deleted.

In one embodiment, the mouse manufactures a light chain comprising a somatically mutated Vl domain obtained from the human Vl gene segment. In one embodiment, the light chain comprises a mutated Vl domain somatically obtained from the human Vl gene segment, and a mouse Ck region. In one embodiment, the mouse does not express a light A string.

In one embodiment, the genetically modified mouse is capable of producing somatic hypermutation of the sequence of the human VL region.

In one embodiment, the mouse comprises a cell that expresses a light chain comprising a mutated human Vl domain somatically linked to a mouse Ck, wherein the light chain is associated with a heavy chain comprising a somatically mutated Vh domain obtained from of a Vh gene segment and wherein the heavy chain comprises a constant region of the mouse heavy chain (Ch). In a specific embodiment, the heavy chain comprises a mouse Ch1, a mouse hinge region, a mouse Ch2, and a mouse Ch3. In a specific embodiment, the heavy chain comprises a human Ch1, a hinge region, a mouse Ch2, and a mouse Ch3.

In one embodiment, the mouse comprises a replacement of endogenous mouse Vh gene segments with one or more human Vh gene segments, wherein the human Vh gene segments are operably linked to a mouse Ch region region, such that the Mouse rearranges the human Vh gene segments and expresses a heavy chain of the reverse chimeric immunoglobulin comprising a human Vh domain and a mouse Ch ch. In one embodiment, 90-100% of the non-rearranged mouse Vh gene segments are replaced with at least one non-rearranged human Vh gene segment. In a specific embodiment, all, or substantially all, of the endogenous mouse Vh gene segments are replaced by at least one non-rearranged Vh gene segment. In one embodiment, the replacement is performed with at least 19, at least 39, or at least 80 or 81 non-rearranged human Vh gene segments. In one embodiment, the replacement is performed with at least 12 functional non-rearranged human Vh gene segments, at least 25 functional non-rearranged human Vh gene segments, or at least 43 functional non-rearranged human Vh gene segments. In one embodiment, the mouse comprises a replacement of all mouse segments Dh and Jh by at least one non-rearranged human Dh segment and at least one non-rearranged human Jh segment. In one embodiment, the at least one non- ^rearranged human Dh segment ^{is selected from 1-1, 1-7, 1-26, 2-8, 2-15, 3-3, 3-10, 3-16, 3- 22, 5-5, 5-12,} 6-6 ^{, 6-13, 7-27, and a} combination thereof. In one embodiment, the at least one non-rearranged human JH segment is selected ^{from 1, 2, 3, 4, 5,} 6 ^{, and a combination thereof. In a specific embodiment, said one, or more,} human Vh ^{gene segment} is selected from 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51, a segment human Vh 6-1 gene, and a combination thereof.

A genetically modified mouse is provided comprising a B lymphocyte that expresses a variable domain of the human light chain (Vl) obtained from a rearranged human Vk 1-39 / Jk sequence that is present in the mouse germ line, where the mouse lacks an endogenous immunoglobulin Vk gene segment and an endogenous non-rearranged immunoglobulin Jk gene segment; and wherein the domain, or domains, human Vl is associated with a variable domain of the human heavy chain (Vh) obtained from a rearranged human Vh / Dh / Jh region selected from 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6-13 / 4. A genetically modified mouse is also provided comprising a B lymphocyte that expresses a human Vl domain obtained from a rearranged human Vk 3-20 / Jk sequence that is present in the mouse germ line, where the mouse lacks a segment endogenous immunoglobulin Vk gene and an endogenous non-rearranged immunoglobulin Jk gene segment; and wherein the domain, or domains, human Vl is associated with a human Vh domain obtained from a rearranged human Vh / Dh / Jh region selected from 3-30 / 3-3 / 3, 3-33 / 1- 7/4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4.

Reference is also made to a mouse comprising a B lymphocyte that expresses a binding protein that specifically binds to an antigen of interest, wherein the binding protein comprises a light chain obtained from a Vk 1-39 rearrangement. / Jk 5 human or a human Vk 3-20 / Jk 1 rearrangement, and wherein the lymphocyte comprises a rearranged immunoglobulin heavy chain gene obtained from a rearrangement of human Vh gene segments selected from a gene segment 1- 69, 2-5, 3-13, 3-23, 3-30, 3-33, 3-53, 4-39, 4-59, and 5-51. In one case, said one or more human Vh gene segments are rearranged with a Jh gene segment ^{of the human heavy chain selected from 1, 2, 3, 4, 5, and} 6 ^{. In one case, said one or more} human ^Vh and Jh ^{gene segments} are rearranged with a human Dh gene segment selected from 1-1, 1-7, 1-26, 2-8, 2-15, 3-3, 3- 10, ^{3-16, 3-22, 5-5, 5-12,} 6-6 ^{, 6-13, and 7-27. In a specific case, the light chain gene has 1,2, 3, 4, or 5 or more} somatic hypermutations.

In one embodiment, the mouse comprises a B lymphocyte comprising a gene sequence of the variable region of the rearranged immunoglobulin heavy chain comprising a Vh / Dh / Jh region selected from 2-5 / 6-6 / 1, 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 3-3 / 4, 3-23 / 3-10 / 4, 3-23 / 6- 6/4, 3-23 / 7-27 / 4, 3-30 / 1 -1/4, 3-30 / 1-7 / 4, 3-30 / 3-3 / 3, 3-30 / 3-3 / 4, 3-30 / 3-22 / 5, 3-30 / 5-5 / 2, 3- 30 / 5-12 / 4, 3-30 / 6-6 / 1, 3-30 / 6-6 / 3, 3-30 / 6-6 / 4, 3-30 / 6-6 / 5, 3-30 / 6-13 / 4, 3-30 / 7-27 / 4, 3-30 / 7- 27/5, 3-30 / 7-27 / 6, 3-33 / 1 -7/4, 3-33 / 2-15 / 4, 4-39 / 1-26 / 3, 4-59 / 3-16 / 3, 4-59 / 3-16 / 4.4-59 / 3- 22/3, 5-51 / 3-16 / 6, 5-51 / 5-5 / 3, 5-51 / 6-13 / 5, 3-53 / 1-1 / 4, 1 -69 / 6-6 / 5, and 1 -69 / 6-13 / 4. In a specific embodiment, the B lymphocyte expresses a binding protein that comprises a variable region of the human immunoglobulin heavy chain fused with a constant region of the mouse heavy chain, and a variable region of the light chain of the Human immunoglobulin fused with a constant region of the mouse light chain. In one embodiment, the rearranged human Vl region is a human Vk1-39Jk5 sequence, and the mouse expresses a reverse chimeric light chain comprising (i) a Vl domain obtained from a Vl / Jl sequence and (ii) a Cl of mouse; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse Ch domain and (ii) a somatically mutated human Vh domain obtained from a human Vh gene segment selected from 1-2, 1- 8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51, a human Vh 6-1 gene segment, and a combination thereof. In one embodiment, the mouse expresses a light chain that is somatically mutated. In one embodiment, the Cl is a mouse Ck. In a specific embodiment, the human Vh gene segment is selected from a 2-5, 3-13, 3-23, 3-30, 4-59, 5-51, and 1-69 gene segment. In a specific embodiment, the somatically mutated human Vh domain comprises a sequence obtained ^{from a Dh segment selected from 1-1, 1-7, 2-8, 3-3, 3-10, 3-16, 3-22 , 5-5, 5-12,} 6-6 ^{, 6-13, and 7-27. In a} specific embodiment, the somatically mutated human Vh domain comprises a sequence obtained from ^{a segment Jh selected from 1, 2, 3, 4, 5, and} 6 ^{. In a specific embodiment, the} somatically ^{mutated human Vh domain} is encoded by a rearranged human Vh / Dh / Jh sequence selected from 2-5 / 6-6 / 1, 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 3-3 / 4, 3-23 / 3-10 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3-30 / 1-7 / 4, 3-30 / 3-3 / 4, 3-30 / 3-22 / 5, 3-30 / 5-5 / 2, 3-30 / 5-12 / 4, 3-30 / 6-6 / 1, 3-30 / 6-6 / 3, 3-30 / 6-6 / 4, 3-30 / 6-6 / 5, 3-30 / 6-13 / 4, 3-30 / 7-27 / 4, 3-30 / 7-27 / 5, 3-30 / 7-27 / 6, 4-59 / 3-16 / 3, 4-59 / 3-16 / 4, 4-59 / 3-22 / 3, 5-51 / 5-5 / 3, 1-69 / 6-6 / 5, and 1-69 / 6-13 / 4.

In one embodiment, the mouse comprises a B lymphocyte that expresses a binding protein that specifically binds to an antigen of interest, wherein the binding protein comprises a light chain obtained from a human Vk1-39 / Jk5 rearrangement, and wherein the lymphocyte comprises a sequence of genes from the heavy chain variable region of the rearranged immunoglobulin comprising a Vh / Dh / Jh region selected from 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6.1-69 / 6-6 / 5 and 1-69 / 6-13 / 4. In a specific embodiment, the B lymphocyte expresses a binding protein that comprises a variable region of the human immunoglobulin heavy chain fused with a constant region of the mouse heavy chain, and a variable region of the human immunoglobulin light chain fused with a constant region of the mouse light chain.

In one embodiment, the human Vl region is a human Vk3-20Jk1 sequence, and the mouse expresses an inverse chimeric light chain comprising (i) a Vl domain obtained from the rearranged human Vl / Jl sequence, and (ii) a Mouse cl; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse Ch, and (ii) a somatically mutated human Vh obtained from a human Vh gene segment selected from 1-2, 1-8, 1 -24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48 , 3-53, 4-31, 4-39, 4-59, 5-51, a human Vh 6-1 gene segment, and a combination thereof. In one embodiment, the mouse expresses a light chain that is somatically mutated. In one embodiment, the Cl is a mouse Ck. In a specific embodiment, the human Vh gene segment is selected from a 3-30, 3-33, 3-53, 4-39, and 51 51 gene segment. In a specific embodiment, the somatically mutated human Vh domain comprises a sequence obtained ^{from a segment Dh selected from} 1-1 ^{, 1-7, 1-26, 2-15, 3-3, 3-16, and 6-13. In a specific embodiment, the} somatically mutated human VH domain comprises a sequence obtained from a JH segment ^{selected from 3, 4, 5, and} 6 ^{. In a specific embodiment, the somatically mutated human VH domain is} encoded by a rearranged human Vh / Dh / Jh sequence selected from 3-30 / 1-1 / 4, 3-30 / 3-3 / 3, 3-33 / 1-7 / 4, 3-33 / 2-15 / 4, 4-39 / 1-26 / 3, 5-51 / 3-16 / 6, 5-51 / 6-13 / 5, and 3-53 / 1-1 / 4.

In one embodiment, the mouse comprises a B lymphocyte that expresses a binding protein that specifically binds to an antigen of interest, wherein the binding protein comprises a light chain obtained from a human Vk3-20 / Jk1 rearrangement, and wherein the lymphocyte comprises a gene sequence of the heavy chain variable region of the rearranged immunoglobulin comprising a Vh / Dh / Jh region selected from 3-30 / 3-3 / 3, 3-33 / 1-7 / 4, 3-33 / 2-15 / 4, and 3-53 / 1-1 / 4. In a specific embodiment, the B lymphocyte expresses a binding protein that comprises a variable region of the human immunoglobulin heavy chain fused with a constant region of the mouse heavy chain, and a variable region of the human immunoglobulin light chain fused with a constant region of the mouse light chain.

In one embodiment, the mouse comprises both a rearranged human Vk1-39Jk5 sequence and a rearranged human Vk3-20Jk1 sequence, and the mouse expresses a reverse chimeric light chain comprising (i) a Vl domain obtained from the sequence Vk1-39Jk5 human or the human Vk3-20Jk1 sequence, and (ii) a mouse Cl; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse Ch, and (ii) a somatically mutated human Vh obtained from a human Vh gene segment selected from 1-2, 1-8 , 1-24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3 -48, 3-53, 4-31, 4-39, 4-59, 5-51, a human Vh 6-1 gene segment, and a combination thereof. In one embodiment, the mouse expresses a light chain that is somatically mutated. In one embodiment, the Cl is a mouse Ck.

In various embodiments, the variable region of the human immunoglobulin heavy chain fused with a constant region of the mouse heavy chain, and the variable region of the human immunoglobulin light chain fused with a constant region of the mouse light chain expressed by lymphocyte B, they are analogous in the mouse. In various embodiments, the chimeric light chain and the chimeric heavy chain expressed by the mouse are analogous in the mouse.

In one embodiment, 90-100% of the rearranged endogenous mouse Vh gene segments are replaced by at least one rearranged human Vh gene segment. In a specific embodiment, all or substantially all non-rearranged endogenous mouse VH gene segments are replaced by at least one non-rearranged human Vh gene segment. In one embodiment, the replacement is performed with at least 18, at least 39, at least 80, or 81 non-rearranged human Vh gene segments. In one embodiment, the replacement is performed with at least 12 functional non-rearranged human Vh gene segments, at least 25 functional non-rearranged human Vh gene segments, or at least 43 non-rearranged human Vh gene segments.

In one embodiment, the genetically modified mouse is a C57BL strain, in a specific embodiment selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, C57BL / O1a. In a specific embodiment, the genetically modified mouse is a mixture of a strain 129 mentioned above and a strain C57BL / 6 mentioned above. In another specific embodiment, the mouse is a mixture of the above-mentioned strains 129, or ^{a mixture of the aforementioned BL} / 6 ^{strains . In a specific embodiment, strain 129 of the mixture} is strain 129S6 (129 / SvEvTac).

In one embodiment, the mouse expresses a reverse chimeric antibody comprising a light chain, which in turn comprises a mouse Ck and a somatically mutated human Vl domain obtained from a rearranged human Vk1-39Jk5 sequence or a Vk3-20Jk1 sequence rearranged human, and a heavy chain comprising a mouse CH and a somatically mutated human VH domain obtained from a human Vh gene segment selected from a human Vh gene segment 1-2, 1-8, 1-24, 1- 69, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3 20, 3-23, 3-30, 3-33, 3-48, 3-53, 4 -31, 4-39, 4-59, 5- 51, and 6-1, wherein the mouse does not express a completely mouse antibody and does not express a completely human antibody. In one embodiment, the mouse comprises a light chain locus k comprising the replacement of mouse endogenous light chain genes segments k by the rearranged human Vk1-39Jk5 sequence or the rearranged human Vk3-20Jk1 sequence, and comprises a rearranged replacement of all, or substantially all, mouse VH gene segments with a complete or substantially complete repertoire of human Vh gene segments.

Reference is also made to a mouse that expresses an immunoglobulin light chain from a sequence of the immunoglobulin light chain rearranged in the mouse germ line, wherein the immunoglobulin light chain comprises a human variable sequence.

In one embodiment, the mouse germ line lacks a gene segment V of the functional non-rearranged immunoglobulin light chain. In one embodiment, the mouse germ line lacks a gene segment J of the functional non-rearranged immunoglobulin light chain.

In one embodiment, the mouse germ line comprises no more than one, no more than two, or no more than three rearranged light chain (V / J) sequences.

The light chain sequence k is a human light chain sequence k. The light chain sequence k is selected from a human Vk1-39 / Jk sequence, a human Vk3-20 / Jk sequence and a combination thereof. In a specific embodiment, the light chain sequence k is a human Vk1 -39 / Jk5 sequence. In a specific embodiment, the light chain sequence k is a human Vk3-20 / Jk1 sequence.

In one embodiment, the mouse further comprises its germ line, a sequence selected from an intronic enhancer mouse k at the 5 'end with respect to the light chain sequence of the rearranged immunoglobulin, a mouse enhancer k in the 3 'end, and a combination thereof.

In one embodiment, the mouse comprises a non-rearranged human Vh gene segment, a non-rearranged human Dh gene segment, and a non-rearranged human Jh gene segment, wherein said Jh gene segments are capable of rearranging to form a variable gene sequence of the immunoglobulin heavy chain operatively linked to a constant heavy chain gene sequence. The mouse comprises a plurality of human Vh, Dh and Jh gene segments. In a specific embodiment, the human Vh, Dh, and Jh gene segments replace the endogenous mouse Vh, Dh, and Jh gene segments in the locus of the endogenous mouse immunoglobulin heavy chain locus. In a specific embodiment, the mouse comprises a replacement of all, or substantially all, of the functional mouse Vh, Dh, and Jh gene segments with all or substantially all functional human Vh, Dh, and Jh gene segments.

In one embodiment, the mouse expresses an immunoglobulin light chain comprising a constant mouse sequence. In one embodiment, the mouse expresses an immunoglobulin light chain comprising a human constant sequence.

In one embodiment, the mouse expresses an immunoglobulin heavy chain comprising a mouse sequence selected from a Ch1 sequence, a hinge sequence, a Ch2 sequence, a Ch3 sequence, and a combination thereof.

In one embodiment, the mouse expresses an immunoglobulin heavy chain comprising a human sequence selected from a Ch1 sequence, a hinge sequence, a Ch2 sequence, a Ch3 sequence, and a combination thereof.

In one embodiment, the sequence of the light chain of the rearranged immunoglobulin in the mouse germ line is in a locus of the light chain of the endogenous mouse immunoglobulin. In a specific embodiment, the sequence of the immunoglobulin light chain rearranged in the mouse germ line replaces all, or substantially all, the V and J sequences of the mouse light chain at the locus of the endogenous immunoglobulin light chain locus. mouse

Reference is also made to a mouse cell, which is isolated from a mouse as described in the present patent. In one example, the cell is an ES cell. In one example, the cell is a lymphocyte. In one example, the lymphocyte is a B lymphocyte. In one example, the B lymphocyte expresses a chimeric heavy chain comprising a variable domain obtained from a human gene segment; and a light chain obtained from a rearranged human Vk1-39 / Jk sequence, a rearranged human Vk3-20 / Jk sequence, or a combination thereof; wherein the variable domain of the heavy chain is fused with a constant mouse region and the variable domain of the light chain is fused with a constant human or mouse region.

Reference is also made to a mouse B lymphocyte that is isolated from a mouse as described in the present patent, wherein the B lymphocyte expresses a chimeric heavy chain obtained from a rearranged human VH / DH / JH sequence selected from between 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4 , 3-30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 and 1-69 / 6-13 / 4; and a chimeric light chain obtained from a rearranged human Vk1-39 / Jk5 sequence; wherein the variable domain is fused with a constant mouse region and the variable domain of the light chain is fused with a constant mouse region.

Reference is also made to a mouse B lymphocyte that is isolated from a mouse as described herein, wherein the B lymphocyte expresses a chimeric heavy chain obtained from a rearranged human VH / DH / JH sequence selected from 3-30 / 3-3 / 3, 3-33 / 1-7 / 4, 3-33 / 2-1 5/4, and 3-53 / 1-1 / 4; and a chimeric light chain obtained from a rearranged human Vk3-20 / Jk1 sequence; wherein the variable domain is fused with a constant mouse region and the variable domain of the light chain is fused with a constant mouse region.

In various examples, the chimeric light and heavy chains expressed by the B-lymphocyte isolated from a mouse, as described herein, are analogous in the mouse.

Reference is also made to a hybridoma, wherein the hybridoma is made with a B lymphocyte of a mouse, as described herein. In a specific example, lymphocyte B is derived from a mouse, as described herein, which has been immunized with an immunogen comprising an epitope of interest, and lymphocyte B expresses a binding protein that binds to the epitope of interest, where the binding protein has a somatically mutated human Vh domain and a mouse Ch domain, and has a human Vl domain obtained from rearranged human Vk1-39Jk5 or rearranged human Vk3-20Jk1 and a mouse Cl.

Reference is also made to a hybridoma that is made with a mouse B lymphocyte, as described in the present patent, wherein the hybridoma expresses a chimeric heavy chain obtained from a rearranged human Vh / Dh / Jh sequence selected from between 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4 , 3-30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 and 1-69 / 6-13 / 4; and a chimeric light chain obtained from a rearranged human Vk1-39 / Jk5 sequence; wherein the variable domain is fused with a constant mouse region and the variable domain of the light chain is fused with a constant mouse region.

Reference is also made to a hybridoma that is made with a mouse B lymphocyte, as described in the present patent, wherein the hybridoma expresses a chimeric heavy chain obtained from a rearranged human Vh / Dh / Jh sequence selected from between 3-30 / 3-3 / 3, 3-33 / 1-7 / 4, 3-33 / 2-15 / 4, and 3-53 / 1-1 / 4; and a chimeric light chain obtained from a rearranged human Vk3-20 / Jk1 sequence; wherein the variable domain is fused with a constant region of a mouse and the variable domain of the light chain is fused with a constant region of mouse.

In various examples, the chimeric light and heavy chains expressed by the hybridoma that is made with a mouse B lymphocyte, as described herein, are analogous in the hybridoma. In various examples, the chimeric light and heavy chains expressed by the hybridoma that is made with a mouse B lymphocyte as described herein are analogous to mouse B lymphocyte.

Reference is also made to a mouse embryo, wherein the embryo comprises a donor ES cell that is obtained from a mouse as described in the present patent.

Reference is also made to an addressing vector, comprising, from the 5 'to 3' end in the transcription direction, in reference to the sequences of the mouse homology arms of the 5 'and 3' end of the vector, a 5 'end mouse homology arm, a human or mouse immunoglobulin promoter, a human or mouse leader sequence, and a human Vl region selected from a rearranged human Vk1-39Jk5 or a Vk3-20Jk1 rearranged human, and a 3 'end homology arm. In one example, the homology arms of the 5 'and 3' end direct the vector to a 5 'end sequence with respect to an enhancer sequence that is present at the 5' end and proximal to the mouse Ck gene. In one example, the promoter is a promoter of a gene segment of the variable region of human immunoglobulin. In a specific example, the promoter is a human Vk3-15 promoter. In one example, the leader sequence is a mouse leader sequence. In a specific example, the mouse leader sequence is a mouse VK3-7 leader sequence.

Reference is also made to an addressing vector as described above, but instead of the homology arm of the 5 'end, the human or mouse promoter is flanked at the 5' end with a site-specific recombinase recognition site. (SRRS), and instead of the mouse homology arm of the 3 'end, the human VL region is flanked at the 3' end with an SRRS.

Reference is also made to a reverse chimeric antibody manufactured by a mouse as described herein, wherein the reverse chimeric antibody comprises a light chain comprising a human Vl and a mouse Cl, and a heavy chain comprising a Vh Human and a mouse Ch.

A method of producing an antibody of interest is also provided comprising a variable domain of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region selected from 2-5 / 3-22 / 1, 3- 13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3- 30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6-13 / 4 associated with a variable region of the human light chain obtained from a rearranged human Vk1-39 / Jk sequence, the method comprising:

(a) immunize the mouse provided with an antigen of interest;

(b) obtain from the mouse a variable domain of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region selected from 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6-13 / 4; Y

(c) employing the sequence of the immunoglobulin variable region obtained in (b) associated with said human light chain variable region in an antibody that specifically binds to the antigen of interest.

Additionally, a method is provided to produce an antibody for an antigen of interest comprising a human heavy chain variable domain obtained from a rearranged human Vh / Dh / Jh region selected from 3-30 / 3-3 / 3 , 3-33 / 1-7 / 4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4 associated with a variable region of the human light chain obtained from a Vk3-20 / Jk sequence rearranged human, understanding the method:

(a) immunize the mouse provided with an antigen of interest;

(b) obtain from the mouse a variable domain of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region selected from 3-30 / 3-3 / 3, 3-33 / 1-7 / 4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4; Y

(c) employing the sequence of the immunoglobulin variable region obtained in (b) associated with said human light chain variable region in an antibody that specifically binds to the antigen of interest.

Reference is also made to a method of making an antibody, which comprises expressing in a cell (a) a first sequence of Vh genes of an immunized mouse as described in the present patent fused with a sequence of Ch genes; (b) a sequence of Vl genes of an immunized mouse as described herein fused with a sequence of human Cl genes; and, (c) keep the cell under conditions sufficient to express a completely human antibody, and isolate the antibody. In one example, the cell comprises a second sequence of a second immunized mouse, as described herein, fused with a sequence of Ch genes, the first sequence of Vh genes encodes a Vh domain that recognizes a first epitope, and The second Vh gene sequence encodes a Vh domain that recognizes a second epitope, where the first epitope and the second epitope are not identical.

Reference is also made to a method for the preparation of an epitope-binding protein, which comprises exposing a mouse such as that described in the present patent to an immunogen comprising an epitope of interest, keeping the mouse under conditions sufficient for the mouse generate an immunoglobulin molecule that specifically binds to the epitope of interest, and isolate the immunoglobulin molecule that specifically binds to the epitope of interest; wherein the epitope binding protein comprises a heavy chain comprising a somatically mutated human Vh and a mouse Ch, associated with a light chain comprising a mouse Cl and a human Vl obtained from rearranged human Vk1-39Jk5 or Human Vk3-20Jk1 reordered.

Reference is also made to a cell expressing an epitope binding protein where the cell comprises: (a) a human nucleotide sequence encoding a human Vl domain that is obtained from a rearranged human Vk1-39Jk5 or a Vk3 -20Jk1 human rearranged, wherein the human nucleotide sequence is fused (directly or through a linker) to a cDNA sequence of a constant domain of the immunoglobulin light chain (for example, a DNA sequence of the constant domain k human); and, (b) a first human Vh nucleotide sequence encoding a Vh domain obtained from a first Vh nucleotide sequence human, wherein the first human Vh nucleotide sequence is fused (directly or through a linker) with a cDNA sequence of a constant domain of the human immunoglobulin heavy chain; wherein the epitope binding protein recognizes a first epitope. In one example, the epitope binding protein ^{binds to the first epitope with a dissociation constant of less than} 10 ^{-6 M, less than} 10 ^{-8 M, less than} 10 ^{-9 M,} less than 10 ^-10 M, less 10 ^-11 M, or less than 10 ^-12 M.

In one example, the cell comprises a second human nucleotide sequence encoding a second human Vh domain, wherein the second human sequence is fused (directly or through a linker) with a cDNA sequence of a heavy chain constant domain of the human immunoglobulin, and where the second human Vh domain does not specifically recognize the first epitope (for example, it shows a dissociation constant of, for example, 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, or greater ), and where the epitope binding protein recognizes the first epitope and the second epitope, and where each, the first and second immunoglobulin heavy chain, is associated with an identical light chain of (a).

In one example, the second Vh domain binds to the second epitope with a dissociation constant that is less than 10 ^-6 M, less than 10 ^-7 M, less than 10 ^-8 M, less than 10 ^-9 M, less than 10 ^-10 M, less than 10 ^-11 M, or less than 10 ^-12 M.

In one example, the epitope binding protein comprises a first immunoglobulin heavy chain and a second immunoglobulin heavy chain, each associated with an identical light chain obtained from a rearranged human Vl region selected from a Vk1- 39Jk5 human or a human Vk3-20Jk1, where the first heavy chain of the immunoglobulin binds to a first epitope with a dissociation constant in a nanomolar to picomolar range, the second heavy chain of the immunoglobulin binds to a second epitope with a dissociation constant in a nanomolar to picomolar range, the first epitope and the second epitope are not identical, the first heavy chain of the immunoglobulin does not bind to the second epitope and binds to the second epitope with a weaker dissociation constant that the micromolar range (for example, the millimolar range), the second heavy chain of the immunoglobulin does not bind to the first epitope or bind to the first epitope with a weaker dissociation constant than the micromolar range (for example, the millimolar range), and one or more of the Vl, the Vh of the first heavy chain of the immunoglobulin, and the VH of the second heavy chain of The immunoglobulin are somatically mutated.

In one example, the first immunoglobulin heavy chain comprises a protein A binding residue, and the second immunoglobulin heavy chain lacks a protein A binding residue.

In one example, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell that expresses a ^{viral nucleic acid sequence (eg, a PERC.} 6 ^{™ cell ).}

Reference is also made to a reverse chimeric antibody comprising a human Vh and a constant domain of the mouse heavy chain, a human Vl and a constant domain of the mouse light chain, wherein the antibody is made by a process comprising immunize a mouse as described herein with an immunogen comprising an epitope, and the antibody specifically binds to the epitope of the immunogen with which the mouse was immunized. In one example, the Vl domain is somatically mutated. In one example, the Vh domain is somatically mutated. In one example, both the Vl domain and the Vh domain are somatically mutated. In one example, the Vl is linked to a mouse Ck domain.

In one aspect, in the provided mouse, the mouse comprises human Vh gene segments that replace all, or substantially all, of the mouse Vh gene segments in the endogenous locus of the mouse heavy chain; where no more than one or two Vl / Jl sequences of the human rearranged light chain selected from a rearranged Vk1-39 / Jk5 and a rearranged Vk3-20 / Jk1 or a combination thereof, replaces all gene segments of the light chain mouse wherein the variable gene segments of the human heavy chain are linked to a mouse constant gene, and the sequences of the rearranged human light chain are linked to a human or mouse constant gene.

Reference is also made to a mouse ES cell comprising a replacement of all or substantially all of the variable gene segments of the mouse heavy chain with variable gene segments of the human heavy chain, and no more than one or two VL / JL sequences of the rearranged human light chain, wherein the variable gene segments of the human heavy chain are linked to a constant gene of the mouse immunoglobulin heavy chain, and the VL / JL sequences of the rearranged human light chain are linked to a constant gene of the human or mouse immunoglobulin light chain. In a specific example, the light chain constant gene is a mouse constant gene.

Reference is also made to an antigen binding protein made by a mouse as described in the present patent. In a specific example, the antigen binding protein comprises a variable region of the human immunoglobulin heavy chain fused with a mouse constant region, and a variable region of the immunoglobulin light chain obtained from a Vk1 gene segment. -39 or a Vk3-20 gene segment ^{, where the constant region of the light chain is a mouse constant region.}

Reference is also made to a fully human antigen binding protein made from a gene sequence of a mouse immunoglobulin variable region as described in the present patent, wherein the antigen binding protein comprises a chain. Fully human weighing comprising a human variable region obtained from a sequence of a mouse as described in the present patent, and a fully human light chain comprising a Vk1-39 or a Vk3-20. In one example, the variable region of the light chain comprises one to five somatic mutations. In one example, the variable region of the light chain is a variable region of the analogous light chain that is paired in a mouse B lymphocyte with the variable region of the heavy chain.

In one example, the fully human antigen binding protein comprises a first heavy chain and a second heavy chain, wherein the first heavy chain and the second heavy chain independently comprise non-identical variable regions obtained from a mouse as described in the present patent, and wherein each of the first and the second heavy chain expresses from a host cell, associated with a human light chain obtained from a Vk1-39 gene segment or a Vk3-20 gene segment. In one example, the first heavy chain comprises a first variable region of the heavy chain that specifically binds to an epitope of a first antigen, and the second heavy chain comprises a second variable region of the heavy chain that specifically binds to a second epitope of a second antigen. In a specific example, the first antigen and the second antigen are different. In a specific example, the first antigen and the second antigen are the same, and the first epitope and the second epitope are not identical; In a specific example, the binding of the first epitope by a first molecule of the binding protein does not block the binding of the second epitope by a second molecule of the binding protein.

In one example, a fully human binding protein, obtained from a human immunoglobulin sequence of a mouse as described herein, comprises a first immunoglobulin heavy chain and a second immunoglobulin heavy chain, wherein the first immunoglobulin heavy chain comprises a first variable region that is not identical to a variable region of the second immunoglobulin heavy chain, and wherein the first immunoglobulin heavy chain comprises a wild-type protein A binding determinant, and the second Heavy chain lacks a wild-type protein A binding determinant. In one example, the first immunoglobulin heavy chain binds to protein A under isolation conditions, and the second immunoglobulin heavy chain does not bind protein A, or it binds protein A at a junction at least 10. times, a hundred times, or a thousand times weaker than the binding of the first heavy chain to protein A under isolation. In a specific example, the first and the second chain are IgG1 isotypes, wherein the second heavy chain comprises a modification selected from 95R (EU 435R), 96F (EU 436F), and a combination thereof, and wherein the First heavy chain lacks such modification.

Reference is also made to a method for making a bispecific antigen binding protein, which comprises exposing a first mouse as described herein to a first antigen of interest comprising a first epitope, exposing a second mouse as describes in the present patent a second antigen of interest comprising a second epitope, allowing the first and second mouse, each, to mount immune responses to the antigens of interest, identify in the first mouse a first variable region of the chain human heavy that binds to the first epitope of the first antigen of interest, identify in the second mouse a second variable region of the human heavy chain that binds to the second epitope of the second antigen of interest, develop a completely human heavy chain gene that encode a first heavy chain that binds to the first epitope of the first antigen of interest, develop a second d gene The fully human heavy chain encoding a second heavy chain that binds to the second epitope of the second antigen of interest, expressing the first heavy chain and the second heavy chain in a cell expressing a single completely human light chain obtained from a segment human Vk1-39 gene or a human Vk3-20 gene segment to form a bispecific antigen binding protein, and isolating the bispecific antigen binding protein.

In one example, the first antigen and the second antigen are not identical.

In one example, the first antigen and the second antigen are identical, and the first epitope and the second epitope are not identical. In one example, the binding of the first variable region of the heavy chain to the first epitope does not block the binding of the second variable region of the heavy chain to the second epitope.

In one example, the first antigen is selected from a soluble antigen and a cell surface antigen (for example, a tumor antigen), and the second antigen comprises a cell surface receptor. In one example, the cell surface receptor is an immunoglobulin receptor. In a specific example, the immunoglobulin receptor is an Fc receptor. In one example, the first antigen and the second antigen are the same cell surface receptor, and the binding of the first heavy chain to the first epitope does not block the binding of the second heavy chain to the second epitope.

In one example, the variable domain of the light chain comprises 2 to 5 somatic mutations. In one example, the variable domain of the light chain is a somatic mutated analogous light chain expressed in a B lymphocyte of the first or second immunized mouse, either with the first or the second variable domain in the chain heavy

In one example, the first fully human heavy chain carries an amino acid modification that reduces its affinity with protein A, and the second fully human heavy chain does not comprise a modification that reduces its affinity with protein A.

Reference is also made to an antibody or a bispecific antibody comprising a variable domain of the human heavy chain made in accordance with the invention. Reference is also made to the use of a mouse as described herein to make a fully human antibody or a completely human bispecific antibody.

In one example, a genetically modified mouse, cell, or embryo described in the present patent comprises a light chain locus k that maintains endogenous regulatory or control elements, for example, a mouse intronic enhancer k, an end enhancer 3 'k of mouse, or both an intronic enhancer and an enhancer of the 3' end, where the regulatory or control elements facilitate somatic mutation or maturation of the affinity of an expressed sequence of the light chain locus k.

Reference is also made to a mouse comprising a population of B lymphocytes characterized by having light immunoglobulin chains obtained from no more than one, or no more than two, gene segments V and J of the rearranged immunoglobulin light chain or non-rearranged, wherein the mouse shows a ratio of the light chain k: A that is approximately the same as that of a mouse comprising a wild type component of the gene segments V and J of the immunoglobulin light chain.

In one example, immunoglobulin light chains are obtained from no more than one, or no more than two gene segments V and J of the immunoglobulin light chain. In a specific embodiment, light chains are obtained from no more than one gene segment V and J of the rearranged immunoglobulin light chain.

In one example, the mouse shows a ratio of the light chain k: A which is approximately 55: 1 to 75: 1, 60: 1 to ^{70: 1.63: 1 to} 68 ^{: 1, or approximately 65: 1 at 67: 1 compared to a mouse comprising a} wild-type ^complement of gene segments V and J of the immunoglobulin light chain. In a specific example, the mouse shows a ratio of the light chain k: A that is 66: 1 compared to a mouse comprising a wild-type complement of the gene segments V and J of the immunoglobulin light chain. In one example, the immunoglobulin light chains obtained from no more than one, or no more than two V and J gene segments of the rearranged or non-rearranged immunoglobulin light chain, include the selected human Vk and J gene segments. of human Vk1-39, human Vk3-20, Jk1 and human Jk5. In a specific example, immunoglobulin light chains are obtained from a single human light chain sequence comprising a human Vk1-39 sequence.

In one example, the mouse shows a ratio of the light chain k: A that is about 18: 1 to 23: 1 or about 19: 1 to 22: 1, compared to a mouse comprising a wild type complement of the V and J gene segments of the immunoglobulin light chain. In one example, the mouse shows a ratio of the light chain k: A that is 21: 1, compared to a mouse comprising a wild-type complement of the gene segments V and J of the immunoglobulin light chain. In one example, the mouse shows a ratio of the light chain k: A that is approximately the same or 20: 1, compared to a mouse comprising a wild type complement of the gene segments V and J of the light chain of immunoglobulin. In one example, the immunoglobulin light chains obtained from no more than one, or no more than two V and J gene segments of the rearranged or non-rearranged immunoglobulin light chain, include human Vk and Jk gene segments selected from Vk1-39 human, Vk3-20 human, Jk1 human and Jk5 human. In a specific example, immunoglobulin light chains are obtained from a single human light chain sequence comprising a human Vk3-20 sequence.

Reference is also made to a mouse that expresses an immunoglobulin light chain obtained from no more than one, or no more than two human Vk / Jk sequences, wherein the mouse comprises a replacement of all or substantially all gene segments. of the variable region of the human heavy chain, and the mouse shows a ratio of (a) CD19 + B lymphocytes expressing an immunoglobulin with a light chain A, with respect to (b) CD19 + B lymphocytes expressing an immunoglobulin with a light chain k, from about 1 to ^about 20 ^.

In one example, the mouse expresses a single light chain k obtained from a human Vk1-39Jk5 sequence, and the ratio of CD19 + B lymphocytes expressing an immunoglobulin with a light A chain with respect to CD19 + B lymphocytes expressing an immunoglobulin with a light chain k is from about 1 to ^about 20 ^{; In one example, the ratio is from about} 1 ^{to at least about} 66 ^{; In a specific embodiment, the ratio is about} 1 ^to 66 ^.

In one example, the mouse expresses a single light chain obtained from a human Vk3-20Jk5 sequence, and the ratio of CD19 + B lymphocytes that express an immunoglobulin with a light chain A relative to B lymphocytes. CD19 + expressing an immunoglobulin with a light chain k is about 1 to about 20; In one example, the ratio is about 1 to about 21. In specific embodiments, the ratio ^is 1 ^to 20 ^{, or} 1 ^to 21 ^.

Reference is also made to a genetically modified mouse that expresses a single rearranged k light chain, wherein the mouse comprises a functional light chain A locus, and where the mouse expresses a population of B lymphocytes comprising IgK + cells that express a light chain k obtained from the same single light chain k rearranged. In one example, the percentage of B lymphocytes with IgK + IgA + in the mouse is approximately the same as in a wild-type mouse. In a specific example, the percentage of B lymphocytes with IgK + IgA + in the ^{mouse is about 2 to about} 6 ^{percent. In a specific example, the percentage of} B lymphocytes with IgK + IgA + in a mouse where the only rearranged light chain k is obtained from a rearranged Vk1-39Jk5 sequence, is from about 2 to about 3; In a specific example, approximately 2.6. In a specific embodiment, the percentage of B lymphocytes with IgK + IgA + in a mouse where the only ^{rearranged light} chain ^{k is obtained from a Vk3-20Jk1 sequence is from about 4 to about} 8 ^{; in a specific embodiment, about} 6 ^.

Reference is also made to a genetically modified mouse, where the mouse expresses a single rearranged light chain k obtained from a human Vk and Jk gene segment, wherein the mouse expresses a population of B lymphocytes comprising a single light chain k obtained from the sequence of the only rearranged k light chain, where the genetically modified mouse has not become resistant to somatic hypermutations. In one embodiment, at least 90% of the light chains k expressed in a mouse B lymphocyte show from at least one to about five somatic hypermutations.

Reference is also made to a genetically modified mouse that is modified to express a single light chain k obtained from no more than one, or no more than two sequences of the rearranged light chain k, where the mouse shows a use of the light chain k which is approximately twice or more, at least three times or more, or at least approximately four or more times greater than the use of the light chain k shown by a wild-type mouse, or greater than the use of the light chain k shown by a mouse of the same strain comprising a repertoire of the gene segments of the light chain k. In a specific example, the mouse expresses the light chain k from no more than one sequence of the rearranged light chain k. In a more specific example, the sequence of the rearranged light chain k is selected from a sequence Vk1-39Jk5 and Vk3-20Jk1. In one example, the sequence of the rearranged light chain k is a sequence Vk1-39Jk5. In one example, the light chain sequence k is a sequence Vk3-20Jk1.

Reference is also made to a genetically modified mouse that expresses a single light chain k obtained from no more than one, or no more than two sequences of the rearranged light chain k, wherein the mouse shows a ^{use of the light chain k which is about} 100 ^{times or more, at least about} 200 ^{times or more,} at least about 300 times or more, at least about 400 times or more, at least about 500 times or more, at least about 600 times or more, at least approximately 700 times or more, at least approximately 800 times or more, at least approximately 900 times or more, at least ^{approximately} 1000 ^{or more times greater than the same use of the same light chain k shown by a mouse} carrying a locus of the complete or substantially complete human light chain k. In a specific example, the mouse carrying a complete or substantially complete human light chain locus k lacks a functional non-rearranged mouse light chain sequence k. In a specific example, the mouse expresses the only light chain ka starting from no more than one sequence of the rearranged light chain k. In one example, the mouse comprises a copy of a sequence of the rearranged light chain k (for example, a heterozygous). In one embodiment, the mouse comprises two copies of a sequence of the rearranged light chain k (eg, a homozygous). In a more specific example, the sequence of the rearranged light chain k is selected from a sequence Vk1-39Jk5 and Vk3-20Jk1. In one example, the sequence of the rearranged light chain k is a sequence Vk1-39Jk5. In one example, the sequence of the rearranged light chain k is a sequence Vk3-20Jk1.

Reference is also made to a genetically modified mouse that expresses a single light chain obtained from no more than one, or no more than two rearranged light chain sequences, wherein the light chain in ^{the genetically modified mouse shows a level of expression that is at least} 10 ^{times to about} 1,000 times, 100 times to about 1,000 times, 200 times to about 1,000 times, 300 times to about 1,000 times, 400 times to about 1,000 times, 500 times to about 1,000 times, 600 times to approximately 1,000 times, 700 times to approximately 1,000 times, 800 times to approximately 1,000 times, or 900 times to approximately 1,000 times higher than the expression of the same rearranged light chain displayed by a mouse which carries a complete or substantially complete light chain locus. In one example, the light chain comprises a human sequence. In a specific example, the human sequence is a k sequence. In one example, the human sequence is a sequence A. In one example, the light chain is a completely human light chain.

In one example, the level of expression is characterized by quantifying the mRNA of the transcribed light chain sequence, and comparing it with the sequence of the transcribed light chain of a mouse carrying a complete or substantially complete light chain locus.

Reference is also made to a genetically modified mouse that expresses a single light chain k obtained from no more than one, or no more than two sequences of the rearranged light chain k, where the mouse, after immunization with the antigen , shows a serum titer that is comparable to a wild-type mouse immunized with the same antigen. In a specific example, the mouse expresses a single light chain k from no more than one sequence of the rearranged light chain k. In one example, the serum titer is characterized as total immunoglobulin. In a specific example, the serum titer is characterized as a specific IgM titer. In a specific example, the serum titer is characterized as a specific IgG titer. In a more specific example, the sequence of the rearranged light chain k is selected from a sequence Vk1-39Jk5 and Vk3-20Jk1. In one example, the sequence of the rearranged light chain k is a sequence Vk1-39Jk5. In one example, the sequence of the rearranged light chain k is a sequence Vk3-20Jk1.

Reference is also made to a genetically modified mouse that expresses a plurality of immunoglobulin heavy chains associated with a single light chain. In one example, the heavy chain comprises a human sequence. In various examples, the human sequence is selected from a variable sequence, a CHi, a hinge region, a CH2, a CH3, and a combination thereof. In one example, the only light chain comprises a human sequence. In various examples, the human sequence is selected from a variable sequence, a constant sequence, and a combination thereof. In one example, the mouse comprises an inactivated endogenous immunoglobulin locus and expresses the heavy chain and / or the light chain from an extrachromosomal transgene or episome. In one example, the mouse comprises a replacement in an endogenous mouse locus of some or all of the endogenous gene segments of the mouse heavy chain (i.e., V, D, J), and / or some or all of the constant sequences endogenous mouse heavy chain (e.g., CHi, hinge region, CH2, CH3, or a combination thereof), and / or some or all of the endogenous mouse light chain sequences (e.g., V, J , constant region, or a combination thereof), with one or more human immunoglobulin sequences.

Reference is also made to a mouse suitable for making antibodies that have the same light chain, wherein all or substantially all antibodies made in the mouse are expressed with the same light chain. In one example, the light chain is expressed from an endogenous light chain locus.

Reference is also made to a method of manufacturing a light chain for a human antibody, which comprises obtaining from a mouse as described in the present patent a light chain sequence and a heavy chain sequence, and which employs the light chain sequence and the heavy chain sequence in the manufacture of a human antibody. In one example, the human antibody is a bispecific antibody.

[Removed]

Brief description of the figures

Fig. 1 illustrates a targeting strategy to replace endogenous gene segments of the variable region of the mouse immunoglobulin light chain with a human Vk1-39Jk5 gene region.

FIG. 2 illustrates a targeting strategy to replace endogenous gene segments of the mouse immunoglobulin light chain variable region with a human Vk3-20Jk1 gene region.

FIG. 3 illustrates a targeting strategy to replace endogenous gene segments of the mouse immunoglobulin light chain variable region with a human VpreB / JA5 gene region.

FIG. 4 shows the percentage of peripheral blood CD19 + B (Y axis) lymphocytes for wild-type mice (WT), homozygous mice for a rearranged human light chain region V k1-39Jk5 (Vk1-39Jk5 HO) engineered and Homozygous mice for a region of the human-engineered Vk3-20Jk1 light chain modified by genetic engineering (Vk3-20Jk1 HO).

FIG. 5A shows the relative expression of the mRNA (y-axis) of a light chain obtained from Vk1-39 in a quantitative PCR assay, using specific probes for linking a region of the human rearranged Vk1-39Jk5 light chain genetically engineered ( Link probe Vk1-39Jk5) and the human Vk1-39 gene segment (Vk1-39 probe) in a homozygous mouse for a replacement of the endogenous gene segments Vk and Jk with human Vk and Jk segments (Hk), a mouse wild type (WT), and a mouse heterozygous for a genetically engineered human-modified Vk1-39Jk5 light chain region (Vk1-39Jk5 HET). The signals are normalized to mouse Ck expression. N.D .: not detected.

FIG. 5B shows the relative expression of the mRNA (-y axis) of a light chain obtained from Vk1-39 in a quantitative PCR assay using specific probes for linking a region of the rearranged human light chain V k1-39Jk5 (binding probe Vk1-39Jk5) and the human Vk1-39 gene segment (Vk1-39 probe) in a homozygous mouse for a replacement of the endogenous Vk and Jk gene segments with human Vk and Jk (Hk) gene segments, a wild-type mouse (WT), and a mouse homozygous for a genetically engineered human-modified Vk1-39Jk5 light chain region (Vk1-39Jk5 HO). The signals are normalized to mouse Ck expression.

FIG. 5C shows the relative expression of the mRNA (-y axis) of a light chain obtained from Vk3-20 in a quantitative PCR assay using specific probes for linking a region of the light chain V k3-20Jk1 rearranged human (Vk3-20Jk1 binding probe) and the human Vk3-20 gene segment (Vk3-20 probe) in a homozygous mouse for a replacement of the Vk and Jk gene segments with the Vk and Jk (Hk) gene segments, a wild-type mouse (WT), and a heterozygous (HET) and homozygous (HO) mouse for a region of the human rearranged Vk3-20Jk1 light chain modified by genetic engineering. The signals are normalized to mouse Ck expression.

FIG. 6A shows the IgM (left) and IgG (right) titer in wild-type (WT; N = 2) and homozygous mice for a genetically engineered human-modified Vk1-39Jk5 light chain region (Vk1-39Jk5 HO; N = 2) immunized with p-galactosidase.

FIG. 6B shows the total immunoglobulin titer (IgM, IgG, IgA) in wild-type mice (WT; N = 5) and in homozygous mice for a genetically engineered human-modified Vk3-20Jk1 light chain region (Vk3 -20Jk1 HO; N = 5), immunized with p-galactosidase.

Detailed description

The present invention is not limited to the particular methods, and to the experimental conditions described, since such methods and conditions may vary. It is also to be understood that the terminology used in the present patent is intended to describe only specific embodiments, and is not intended to be limiting, since the scope of the present invention is defined by the claims.

[Removed]

The term "antibody", as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a variable region of the heavy chain (Vh) and a constant region of the heavy chain (Ch). The constant region of the heavy chain comprises three domains, Ch1, Ch2 and Ch3. Each light chain comprises a variable region of the light chain (Vl) and a constant region of the light chain (Cl). Regions Vh and Vl can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), intermingled with regions that are more conserved, called framework regions (FR). Each Vh and Vl comprises three CDR and four FR, arranged from the amino-terminal end to the carboxy-terminal end in the following order: FRl, CDR1, FR2, CDR2, FR3, CDR3, FR4 (the heavy chain CDRs can abbreviated as HCDR1, HCDR2 and HCDR3, the light chain CDRs can be abbreviated as LCDR1, LCDR2 and LCDR3 The term "high affinity" ^antibody refers to ^{an antibody having a Kd with respect to its target epitope of approximately 10} ' 9 ^{M or less (for example, about 1 x} 10'9 ^{M, 1 x} 10'10 ^{M, 1 x} 10'11 ^{M, or about 1 x} 10'12 ^{M). In one embodiment, the} Kd is measured by surface plasmon resonance, for example, from BIACORE ™; in another embodiment, Kd is measured by ELISA.

The phrase "bispecific antibody" includes an antibody capable of selectively binding two or more epitopes. Antibodies generally comprise two non-identical heavy chains, where each heavy chain specifically binds to a different epitope - either in two different molecules (for example, different epitopes in two different immunogens) or in the same molecule (for example, different epitopes in the same immunogen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will be at least one to two or three or four or more orders of magnitude less than the affinity of the first heavy chain for the second epitope, and vice versa. Epitopes specifically bound by the bispecific antibody can be on the same or on a different target (for example, on the same or on a different protein). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same immunogen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same immunogen can be fused to nucleic acid sequences encoding the same or different constant regions of the heavy chain, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain. A typical bispecific antibody has two heavy chains, each with three CDRs of the heavy chain, followed by (from the N-terminal to the C-terminal) a Ch1 domain, a hinge region, a Ch2 domain, and a Ch3 domain, and an immunoglobulin light chain that either does not confer an epitope binding specificity but that can be associated with each heavy chain, or that can be associated with each heavy chain and that can bind to one or more epitopes linked by the binding regions to the epitope of the heavy chain, or that can be associated with each heavy chain and enable the union of one or both heavy chains to one or both epitopes.

The term "cell" includes any cell that is suitable for expressing a sequence of recombinant nucleic acids. Cells include those prokaryotic and eukaryotic (single cell or multi-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., Etc.), mycobacterial cells, fungal cells, cells of yeasts (for example, S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (for example, SF-9, SF-21, insect cells infected by baculovirus , Trichoplusia ni, etc.), cells of non-human animals, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some cases, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some examples, the cell is eukaryotic and is selected from the following cells: CHO (for example, CHO K1, DXB-1 1 CHO, Veggie-CHO), COS (for example, COS-7), retinal cell, Vero, CV1, kidney (for example, HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (for example, BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2 / 0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line obtained from a cell mentioned above. In some embodiments, the cell comprises one or more viral genes, for example, a ^{retinal cell that expresses a viral gene (for example, a PER.C} 6 ^{™ cell ).}

The phrase "complementarity determining region", or the term "CDR" includes an amino acid sequence encoded by a nucleic acid sequence of the immunoglobulin genes of an organism that normally (ie, in a wild-type animal) appears between two framework regions in a variable region of a light or heavy chain of an immunoglobulin molecule (for example, an antibody or a T-cell receptor). A CDR can be encoded by, for example, a germline sequence or a rearranged or non-rearranged sequence, and, for example, by a B lymphocyte or a naive or mature T lymphocyte. A CDR can be somatically mutated (for example, varying from a sequence encoded in an animal's germ line), humanized, and / or modified with amino acid substitutions, additions, or deletions. In some circumstances (for example, for a CDR3), the CDRs can be encoded by two or more sequences (for example, germline sequences) that are not contiguous (for example, in a non-rearranged nucleic acid sequence), but they are contiguous in a B-cell nucleic acid sequence, for example, as a result of splicing or connection of the sequences (eg, recombination of VDJ to form a heavy chain c DR3).

The term "conservative" when used to describe a conservative amino acid substitution, includes the substitution of an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (eg, loading or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of a variable region to specifically bind a target epitope with a desired affinity. Examples of amino acid groups having side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic hydroxyl side chains such as serine and threonine; side chains containing amide such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine. Conservative amino acid substitution groups include, for example, valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamate / aspartate, and asparagine / glutamine. In some embodiments, a conservative amino acid substitution may be a substitution of any native residue in a protein with alanine, as used in, for example, alanine scan mutagenesis. In some embodiments, a conservative substitution is made that has a positive value in the logarithmic likelihood matrix PAM250 disclosed in Gonnet et al. (1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256: 1443-45. In some embodiments, the substitution is a moderately conservative substitution where the substitution has a non-negative value in the logarithmic likelihood matrix PAM250.

In some examples, the positions of residues in a light or heavy chain of immunoglobulin differ in one or more conservative amino acid substitutions. In some embodiments, the positions of the residues in an immunoglobulin light chain or a functional fragment thereof (eg, a fragment that allows expression and secretion from, for example, a B lymphocyte) are not identical to a light chain whose amino acid sequence is detailed in the present patent, but differs in one or more conservative amino acid substitutions.

The phrase "epitope binding protein" includes a protein that has at least one CDR and that is capable of recognizing an epitope selectively, for example is capable of binding to an epitope with a Kd that is of a micromolar or ^{less ( for example, a Kd that is approximately 1 x 10} '6 ^{M, 1 x 10'} 7 ^{M, 1 x 10} '9 ^{M, 1 x 10'} 9 ^{M, 1 x 10} '10 ^{M, 1 x} 10'11 ^{M , or about} 1 ^x 10'12 ^{M). Therapeutic epitope binding proteins (for example,} therapeutic ^antibodies ) frequently require a Kd that is in the nanomolar or picomolar range.

The phrase "functional fragment" includes epitope-binding protein fragments that can be expressed, secreted and specifically bound to an epitope with a Kd in the micromolar, nanomolar, or picomolar range. Specific recognition includes having a Kd that is at least in the micromolar range, the nanomolar range, or the picomolar range.

The term "germ line" includes reference to an immunoglobulin nucleic acid sequence in a non-somatic mutated cell, for example, a non-somatic mutated B lymphocyte or a pre-B lymphocyte or a hematopoietic cell.

The phrase "heavy chain", or "immunoglobulin heavy chain" includes a sequence of the constant region of the immunoglobulin heavy chain of any organism. Variable domains of the heavy chain include three CDRs and four FR regions, unless otherwise specified. Heavy chain fragments include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has, following the variable domain (from the N-terminal end to the C-terminal end), a Ch1 domain, a hinge region, a domain Ch2, and a Ch3 domain. A functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an epitope (for example, recognizing the epitope with a Kd in the micromolar, nanomolar, or picomolar range), which is capable of expressing and secreting from of a cell, and that comprises at least one CDR.

The term "identity" when used in connection with a sequence includes identity as determined by a number of different algorithms known in the art that can be used to measure the identity of a nucleotide and / or an amino acid sequence. In some embodiments described in this patent, identities are determined using an alignment with ClustalW v. 1.83 (slow) using a gap ^opening penalty ^{(gaps) of} 10 ^, 0 ^{, a gap extension penalty of} 0 ^, 1 ^{, and using a} Gonnet ^{similarity matrix} (MACVECTOR ™ 10.0.2 program, MacVector Inc. , 2008). The length of the sequences compared to the sequence identity will depend on the particular sequences, but in the case of a constant domain of the light chain, the length should contain a sequence of sufficient length to fold into a domain of the light chain that is capable of self-association to form a constant canonical domain of the light chain, for example, capable of forming two beta sheets comprising beta strands (conformations) and that are capable of interacting with at least one Ch1 domain of A human or a mouse. In the case of a Ch1 domain, the sequence length should contain a sequence of sufficient length to fold into a CH1 domain that is capable of forming two beta sheets comprising beta strands and that are capable of interacting with at least one constant domain. of the light chain of a mouse or a human.

The phrase "immunoglobulin molecule" includes two heavy chains of immunoglobulin and two light chains of immunoglobulin. Heavy chains may be identical or different, and light chains may be identical or different.

The phrase "light chain" includes a sequence of the immunoglobulin light chain of any organism, and unless otherwise specified, includes human k and A light chains and a VpreB, in addition to substitution light chains. Variable domains of the light chain (Vl) usually include three CDRs of the light chain and four framework regions (FR), unless otherwise specified. Generally, a full length light chain includes, from the amino-terminal to the carboxyl-terminal end, a Vl domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a constant light chain domain . Light chains include, for example, those that do not selectively bind to a first or second epitope selectively linked by the epitope binding protein in which they appear. Light chains also include those that bind and recognize, or help the heavy chain to bind to and recognize, one or more epitopes selectively linked by the epitope binding protein in which they appear. Common light chains are those obtained from a rearranged human Vk1-39Jk5 sequence or a rearranged human Vk3-20Jk1 sequence, and include somatically mutated versions (eg, with affinity maturation).

The phrase "micromolar range" is intended to mean a range of 1-999 micromolar; the phrase "nanomolar range" is intended to mean a range of 1-999 nanomolar; The phrase "picomolar range" is intended to mean a range of 1-999 picomolar.

The phrase "somatically mutated" includes the reference to a nucleic acid sequence of a B lymphocyte that has undergone a class switching, wherein the nucleic acid sequence of a variable region of the immunoglobulin (eg, a variable domain of the heavy chain or that includes a sequence of the CDR or FR of the heavy chain) in the B-switched class B is not identical to the nucleic acid sequence in the B-cell before class switching, such as, for example , a difference in a CDR or a nucleic acid sequence of a framework region between a B lymphocyte that has not undergone class switching and a B lymphocyte that has undergone class switching. "Somatically mutated" includes the reference to nucleic acid sequences of B lymphocytes with affinity maturation that are not identical to corresponding immunoglobulin variable region sequences, in B lymphocytes that do not have affinity maturation (ie, sequences in the genome of germline cells). The phrase "somatically mutated" further includes reference to a nucleic acid sequence of the immunoglobulin variable region of a B lymphocyte after exposure of the B lymphocyte to an epitope of interest, wherein the nucleic acid sequence differs from the corresponding nucleic acid sequence prior to exposure of the B lymphocyte to the epitope of interest. The phrase "somatically mutated" refers to antibody sequences that have been generated in an animal, for example, a mouse having nucleic acid sequences of the variable region of the human immunoglobulin, in response to an immunogen exposure test. , and that is the result of the inherently operative selection processes in said animal.

The term "non-rearranged", in reference to a nucleic acid sequence, includes nucleic acid sequences that exist in the germ line of an animal cell.

The phrase "variable domain" includes an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) comprising the following amino acid regions from the N-terminal to the C-terminal (unless indicated by other mode): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Common light chain

Previous efforts to make multispecific epitope binding proteins useful, for example, bispecific antibodies, have been hampered by a variety of problems that frequently share a common paradigm: in vitro selection or manipulation of sequences to be engineered by genetic engineering. Rational form, or to be engineered by trial and error, a suitable format to match a heterodimeric bispecific human immunoglobulin. Unfortunately, most, if not all, in vitro genetic engineering approaches provide ad hoc corrections that are largely suitable, in any case, for individual molecules. On the other hand, no methods have been performed in vivo to employ complex organisms to select appropriate pairings that are capable of leading to therapeutics in humans.

In general, native mouse sequences are often not a good source for human therapeutic sequences. For at least that reason, generating variable regions of the heavy chain immunoglobulin that pairs with a common light chain is of limited practical utility. More efforts would be consumed in vitro in a trial and error process to try to humanize the variable sequences of the mouse heavy chain, while waiting to maintain the specificity and affinity of the epitope while maintaining the ability to attach to the light chain common human, with an uncertain result. At the end of that process, the final product can maintain some specificity, and be associated with the common light chain, but ultimately immunogenicity in a human would probably continue to be a profound risk.

Therefore, a mouse suitable for developing a human therapy would include a suitably large repertoire of gene segments of the human heavy chain variable region, rather than endogenous variable gene segments of the mouse heavy chain. The gene segments of the variable region of the human heavy chain should be able to be rearranged and recombined with an endogenous constant domain of the mouse heavy chain to form an inverse chimeric heavy chain (i.e., a heavy chain comprising a human variable domain and a constant mouse region). The heavy chain should be capable of class switching and hypermutation so that a suitably large repertoire of the variable domains of the heavy chain is available so that the mouse selects one that can be associated with the limited repertoire of the variable regions of the light chain human

A mouse that selects a common light chain for a plurality of heavy chains has practical utility. In various embodiments, antibodies that express in a mouse that can only express a common light chain will have heavy chains that can be associated and expressed with an identical or substantially identical light chain. This is particularly useful in the development of bispecific antibodies. For example, such a mouse can be immunized with a first immunogen to generate a B lymphocyte that expresses an antibody that specifically binds to a first epitope. The mouse (or a genetically equal mouse) can be immunized with a second immunogen to generate a B lymphocyte that expresses an antibody that specifically binds to the second epitope. The variable regions can be cloned from B lymphocytes and expressed with the same constant region of the heavy chain, and the same light chain, and expressed in a cell to make a bispecific antibody, wherein the light chain component of the bispecific antibody It has been selected by a mouse to associate and express with the light chain component.

The inventors have genetically engineered a mouse to generate immunoglobulin light chains that will properly pair with a fairly diverse family of heavy chains, including heavy chains whose variable regions move away from the germline sequences, for example regions variables with affinity maturity or somatically mutated. In various embodiments, the mouse is designed to match variable domains of the human light chain with variable domains of the human heavy chain comprising somatic mutations, thereby enabling a route to high affinity binding proteins suitable for use as a therapeutic in humans.

The genetically engineered mouse, through the long and complex processes of antibody selection inside an organism, makes biologically appropriate choices when matching a diverse collection of variable domains of the human heavy chain with a number Limited human light chain options. To achieve this, the mouse is genetically engineered to present a limited number of variable domain options of the human light chain, in conjunction with a wide variety of variable domain options of the human heavy chain. Following a challenge test with an immunogen, the mouse maximizes the number of solutions in its repertoire to develop an antibody to the immunogen, largely limited or only by the number of light chain options in its repertoire. In various embodiments, this includes allowing the mouse to achieve adequate and compatible mutations of the variable domain of the light chain that will, however, be compatible with a relatively large variety of variable domains of the human heavy chain, including in particular variable domains of the Human heavy chain mutated somatically.

To achieve a limited repertoire of light chain options, the mouse is engineered to become non-functional or substantially non-functional, its ability to develop, or reorder, a native variable domain of the mouse light chain. This can be achieved, for example, by deletion of the gene segments of the mouse light chain variable region. The endogenous mouse locus can then be modified by an exogenous gene segment of the variable region of the human light chain of choice, operatively linked to the endogenous constant domain of the mouse light chain, such that the exogenous gene segments of the human variable region can combine with the endogenous gene of the mouse light chain constant region and form a reverse chimeric light chain gene rearranged (human variable, mouse constant). In various embodiments, the variable region of the light chain can be somatically mutated. In various embodiments, to maximize the ability of the light chain variable region to acquire somatic mutations, the appropriate enhancer or enhancers are maintained in the mouse. For example, when modifying a locus of the mouse light chain k to replace endogenous gene segments of the mouse light chain k with gene segments of the human light chain k, the mouse intronic enhancer k and the enhancer 3 ' mouse are maintained or not functionally altered.

A genetically engineered mouse is provided that expresses a limited repertoire of reverse chimeric light chains (human variable, mouse constant) associated with a variety of reverse chimeric heavy chains (human variable, mouse constant). In various embodiments, the endogenous gene segments of the mouse k light chain are removed and replaced with a single (or two) region of the rearranged human light chain, operably linked to the endogenous mouse Ck gene. In embodiments to maximize somatic hypermutation of the rearranged light chain region, the mouse intronic enhancer k and the mouse 3 'enhancer are maintained. In various embodiments, the mouse also comprises a non-functional light chain A locus, or a deletion thereof or a deletion that makes the locus unable to manufacture a light chain A.

Therefore, a genetically modified mouse comprising a B lymphocyte expressing a variable region (Vl) domain of the human light chain obtained from a rearranged human Vk1-39 / Jk sequence that is present in the line is provided germinal of the mouse, where the mouse lacks an endogenous non-rearranged immunoglobulin Vk gene segment, and a non-rearranged immunoglobulin Jk endogenous segment; and wherein the human domain, or domains, Vl is associated with a variable domain (Vh) of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region selected from 2-5 / 3-22 / 1 , 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4 , 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6-13 / 4 . Also provided is a genetically modified mouse comprising a B lymphocyte that expresses a variable domain of the human light chain (Vl) obtained from a rearranged human Vk3-20 / Jk sequence that is present in the mouse germ line, where the mouse lacks an endogenous non-rearranged immunoglobulin Vk gene segment and a non-rearranged immunoglobulin Jk gene segment; and wherein the domain, or domains, Vl is associated with a variable domain (Vh) of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region selected from 3-30 / 3-3 / 3, 3-33 / 1-7 / 4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4.

The genetically engineered mouse provided in various embodiments, comprises a locus of the variable region of the light chain that lacks the endogenous gene segments Vl and Jl of the mouse light chain, and which comprises a variable region of the human light chain rearranged, which is a rearranged human Vk1-39 / Jk sequence or a human Vk3-20 / Jk sequence, operatively linked to a constant mouse region, where the locus is capable of somatic hypermutation, and where the locus expresses a light chain comprising the Vl / Jl sequence linked to a mouse constant region. Thus, in various embodiments, the locus comprises a mouse 3 k enhancer, which is correlated with a normal or wild-type somatic hypermutation level.

The genetically engineered mouse, in various embodiments when immunized with an antigen of interest generates B lymphocytes that show a variety of rearrangements of the variable regions of the human immunoglobulin heavy chain that express and function with one or two light chains reordered, including embodiments in which said one or two light chains comprise variable regions of the human light chain comprising, for example, 1 to 5 somatic mutations. In various embodiments, human light chains expressed in this way are capable of associating and expressing with any variable region of the immunoglobulin heavy chain expressed in the mouse. In one example, the genetically modified mouse comprises a B lymphocyte that expresses a human Vl domain obtained from a rearranged human Vk1-39 / Jk sequence that is present in the mouse germ line, where the mouse lacks a gene segment Vk endogenous non-rearranged immunoglobulin and an endogenous Jk gene segment non-rearranged immunoglobulin; and wherein the domain, or domains, variable (Vl) of the human light chain is associated with a variable domain (Vh) of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region selected from 2- 5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3- 30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1- 69 / 6-13 / 4. In another example, the genetically modified mouse comprises a B lymphocyte that expresses a variable domain (VL) of the human light chain obtained from a rearranged human Vk3-20 / Jk sequence that is present in the mouse germ line, where the mouse lacks an endogenous Vk gene segment of the non-rearranged immunoglobulin and an endogenous Jk gene segment of the non-rearranged immunoglobulin; and wherein the domain, or domains, human VL is associated with a variable domain (VH) of the human heavy chain obtained from a human rearranged Vh / Dh / Jh region selected from 3-30 / 3-3 / 3 , 3-33 / 1-7 / 4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4.

Epitope binding proteins that bind to more than one epitope

The compositions and methods described in the present patent can be used to make binding proteins that bind to more than one epitope with a high affinity, for example, bispecific antibodies. Among the advantages of The invention includes the ability to select heavy chain immunoglobulin chains with suitably high binding (eg, with affinity maturation), each of which will be associated with a single light chain.

The synthesis and expression of bispecific binding proteins has been problematic, partly due to factors associated with the identification of a suitable light chain that can be associated and expressed with two different heavy chains, and partly due to isolation factors. The methods and compositions described in this patent allow a genetically modified mouse to select, through otherwise natural processes, a suitable light chain that can be associated and expressed with more than one heavy chain, including heavy chains that are somatically mutated ( for example, with maturity of affinity). Human Vl and Vh sequences of suitable B lymphocytes from immunized mice as described herein, which express affinity maturity antibodies that have reverse chimeric heavy chains (i.e., human variable and mouse constant), can be identified. and cloned into a frame in an expression vector with a sequence of genes from the appropriate human constant region (eg, a human IgG1). Two such constructs can be prepared, where each construct encodes a variable domain of the human heavy chain that binds to a different epitope. One of the human Vl (for example, human Vk1-39Jk5 or human Vk3-20Jk1), in the germline sequence or of a B lymphocyte where the sequence has been somatically mutated, can be fused in a gene frame of the appropriate human constant region (for example, a human constant gene k). These three light and heavy all-human constructs can be placed in a cell suitable for expression. The cell will express two main species: a homodimeric heavy chain with the identical light chain, and a heterodimeric heavy chain with the identical light chain. To allow easy separation of these major species, one of the heavy chains is modified to omit a binding determinant to protein A, which results in a differential affinity of a homodimeric binding protein, of a heterodimeric binding protein. The compositions and methods that address this issue are described in USSN 12 / 832,838, filed on June 25, 2010, entitled "Readily Isolated Bispecific Antibodies with Native Immunoglobulin Format," published as US 2010 / 0331527A1.

In one example, reference is made to an epitope binding protein as described in the present patent, wherein the human sequences Vl and Vh are obtained from mice described in the present patent that have been immunized with an antigen comprising An epitope of interest.

Reference is also made to an epitope binding protein comprising a first and a second polypeptide, wherein the first polypeptide comprises, from the N-terminal end to the C-terminal end, a first binding region to a first epitope that binds selectively a first epitope, followed by a constant region comprising a first Ch3 region of a human IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and a second polypeptide comprising, from the N-terminal to the C-terminal end, a second epitope binding region that selectively binds to a second epitope, followed by a constant region comprising a second Ch3 region of a selected human IgG between IgG1, IgG2, IgG4, and a combination thereof, wherein the second Ch3 region comprises a modification that reduces or eliminates the binding of the second Ch3 domain to protein A.

In one example, the second region Ch3 comprises a modification H95R (according to IMGT exon numbering; H435R according to EU numbering). In another embodiment, the second region Ch3 further comprises a Y96F modification (IMGT; Y436F of the EU).

In one example, the second Ch3 region is of a modified human IgG1, and also comprises a modification selected from the group consisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L358M, N384S, K392N , V397M, and EU V422I).

In one example, the second Ch3 region is of a modified human IgG2, and further comprises a modification selected from the group consisting of N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I of the EU).

In one example, the second Ch3 region is of a modified human IgG4, and also comprises a modification selected from the group consisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT; Q355R, N384S, K392N , V397M, R409K, E419Q, and EU V4221).

A method referred to herein to make an epitope binding protein that binds to more than one epitope, is to immunize a first mouse according to the invention an antigen comprising a first epitope of interest, in where the mouse comprises an endogenous locus of the variable region of the immunoglobulin light chain that does not contain an endogenous mouse Vl that is capable of rearranging and forming a light chain, where in the endogenous locus of the variable region of the chain Lightweight of the mouse immunoglobulin is a unique rearranged human Vl region operatively linked to the endogenous gene of the constant region of the mouse light chain, and the rearranged human Vl region is selected from a human Vk1-39Jk5 and a human Vk3-20Jk1 , and mouse endogenous Vh gene segments have been replaced in whole or in part with human Vh gene segments, such that heavy chains of immunoglobulin made by the mouse are only or substantially heavy chains comprising human variable domains and domains mouse constants When immunized, such a mouse will make a reverse chimeric antibody, comprising only one or two variable domains of the human light chain (for example, one of human Vk1-39Jk5 or one of human Vk3-20Jk1). Once it is identified that a B lymphocyte encodes a Vh that binds to the epitope of interest, the nucleotide sequence of Vh (and, optionally, Vl) can be recovered (for example, by PCR) and cloned into a expression construct in a frame with a constant domain of suitable human immunoglobulin. This process can be repeated to identify a second domain that binds to a second epitope, and a second Vh gene sequence can be recovered and cloned into an expression vector in a frame for a second constant domain of suitable immunoglobulin. The first and second constant domain of the immunoglobulin can be an equal or different isotype, and one of the constant domains of the immunoglobulin (but not the other), can be modified as described in this patent or in US 2010 / 0331527A1 , and an epitope binding protein can be expressed in a suitable and isolated cell based on its differential affinity for protein A, compared to a homodimeric epitope binding protein, for example, as described in US 2010. / 0331527A1.

Reference is also made to a method for making a bispecific epitope binding protein, which comprises identifying a first human Vh (Vh1) nucleotide sequence with affinity maturity (for example, comprising one or more somatic hypermutations) of a mouse as described herein, identify a second human VH (VH2) nucleotide sequence with affinity maturity (eg, comprising one or more somatic hypermutations) of a mouse as described herein, clone Vh1 into a frame with a human heavy chain that lacks a determinant modification of protein A, as described in US 2010 / 0331527A1 to form a heavy chain 1 (HC1), clone Vh2 in a frame with a human heavy chain comprising a determinant of Protein A as described in US 2010 / 0331527A1 to form heavy chain 2 (HC2), introduce an expression vector comprising HC1 and the same or a vector of different expression comprising HC2 in a cell, wherein the cell also expresses a light chain of human immunoglobulin comprising a human Vk1-39 human / Jk5 or a human Vk3-20 / human JKl fused with a constant domain of the chain light human, allowing the cell to express a bispecific epitope binding protein comprising a Vh domain encoded by Vh1 and a Vh domain encoded by Vh2, and isolating the bispecific epitope binding protein based on its differential ability to bind to the Protein A compared to a monospecific homodimeric epitope binding protein. In a specific example, HC1 is an IgG1, and HC2 is an IgG1 that comprises modification H95R (IMGT; H435R of the EU), and also comprises modification Y96F (IMGT; Y436f of the EU). In one example, the Vh domain encoded by Vh1, the Vh domain encoded by Vh2, or both, are somatically mutated.

Human VH genes that are expressed with a common human VL

A variety of human variable regions of affinity maturity antibodies created against four different antigens were expressed either with their analogous light chain, or at least one of the selected human light chains of human Vk1-39Jk5, human Vk3-20Jk1, or Human VpreBJA5 (see Example 1). For the antibodies for each of the antigens, heavy chains, somatically mutated and with high affinity of different gene families were successfully matched with the Vk1-39Jk5 and Vk3-20Jk1 regions of the rearranged human germline, and were secreted to from cells that express heavy and light chains. For Vk1-39Jk5 and Vk3-20Jk1, the Vh domains obtained from the following families of human Vh genes were ^favorably expressed ^{: 1-2, 1-8, 1-24, 2-5, 3-7, 3-9 , 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31,4-39, 4-59, 5-51, and} 6 ^­ 1. Therefore, a mouse that is genetically engineered to express a limited repertoire of human VL domains from one or both of Vk1-39Jk5 and Vk3-20Jk1, will generate a diverse population of human Vh domains somatically mutated from a modified VH locus to replace mouse VH gene segments with human VH gene segments.

Genetically engineered mice to express reverse chimeric immunoglobulin heavy chains (human variable, mouse constant), associated with a single rearranged light chain (e.g., a Vk1-39 / J or a Vk3-20 / J), when immunized with an antigen of interest, they generated B lymphocytes that comprised a variety of human Vh rearrangements and expressed a variety of high affinity antigen specific antibodies with various properties with respect to their ability to block the binding of the antigen to its ligand, and with respect to their ability to bind to antigen variants (see Examples 5 to 10).

Therefore, the mice and methods described in the present patent are useful in making and selecting variable domains of the immunoglobulin heavy chain, which include somatically mutated human heavy chain variable domains, which are the result of a diversity of rearrangements, which show a wide variety of affinities (including showing a Kd in approximately nanomolar range or less), a wide variety of specificities (including binding to different epitopes of the same antigen), and that are associated and expressed with the same, or substantially the same variable region of the human immunoglobulin light chain.

The following examples are provided to describe to those skilled in the art how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors consider as their invention. Efforts have been made to ensure accuracy with respect to the numbers used (for example, quantities, temperature, etc.), but some experimental errors and deviations should be considered. Unless stated otherwise, the parts are parts by weight, the molecular weight is average molecular weight, the temperature is indicated in degrees Celsius, and the pressure is, or is close to, atmospheric pressure.

Examples

The following examples are provided to describe how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors consider their invention. Unless stated otherwise, the temperature is indicated in degrees Celsius, and the pressure is, or is close to, atmospheric pressure.

Example 1

Identification of the V ^h regions that are associated with selected human V ^l regions

An in vitro expression system was constructed to determine if a single light chain of the human germ line could be co-expressed with human heavy chains of human antigen-specific antibodies.

Methods for generating human antibodies in genetically modified mice are known (see, for example, US Patent 6,596,541, from Regeneron Pharmaceuticals, VELOCIMMUNE®). The VELo C iMMUNE® technology involves the generation of a genetically modified mouse that has a genome comprising variable regions of the human light and heavy chain operatively linked to endogenous locuses of the mouse constant region, such that the mouse produces an antibody which comprises a human variable region and a mouse constant region in response to antigenic stimulation. The DNA that encodes the variable regions of the light and heavy chains of the antibodies produced from a VELOCIMMUNE® mouse are completely human. Initially, high affinity chimeric antibodies that have a human variable region and a mouse constant region are isolated. As described below, antibodies are characterized and selected to have desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate a completely human antibody that contains a non-IgM isotype, for example, a wild-type or modified IgG1, IgG2, IgG3 or IgG4. While the selected constant region may vary according to a specific use, the antigen binding characteristics with high affinity and the target specificity reside in the variable region.

A VELOCIMMUNE® mouse was immunized with a growth factor that promotes angiogenesis (Antigen C) and antigen-specific human antibodies were isolated and sequenced to determine the use of the V gene, using standard techniques recognized in the art. The selected antibodies were cloned into constant regions of the human light and heavy chain, and 69 heavy chains were selected to pair with one of three human light chains: (1) the analog light chain k linked to a human constant region k, (2 ) a Vk1-39Jk5 of the rearranged human germline linked to a human constant region k, or (3) a Vk3-20Jk1 of the rearranged human germline linked to a human constant region k. Each heavy chain and light chain pair was co-transfected into CHO-K1 cells using standard techniques. The presence of an antibody in the supernatant was detected by an anti-human IgG in an ELISA assay. The antibody titer (ng / ml) was determined for each heavy chain / light chain pair, and the titles with the different light chains of the rearranged germ line were compared with the titers obtained with the parental antibody molecule (i.e., the heavy chain with the analogous light chain), and the percentage of native titer was calculated (Table 1). Vh: heavy chain variable gene. ND: no expression detected under current experimental conditions.

Table 1

continuation

In a similar experiment, VELOCIMMUNE® mice were immunized with several different antigens and the selected antigen-specific human antibody heavy chains were tested for their ability to pair with different light chains of the rearranged human germline (as described above). ). The antigens used in the experiment included an enzyme involved in cholesterol homeostasis (Antigen A), a serum hormone involved in the regulation of glucose homeostasis (Antigen B), a growth factor that promotes angiogenesis (Antigen C) and a cell surface receptor (Antigen D). Antigen-specific antibodies were isolated from mice of each immunization group, and the variable regions of the light chain and heavy chain were cloned and sequenced. From the sequence of the heavy and light chains, the use of the V gene was determined and the selected heavy chains were matched either with its analogous light chain, or with a Vk1-39Jk5 region of the rearranged human germline. Each heavy chain / light chain pair was co-transfected into CHO-K1 cells and the presence of an antibody in the supernatant was detected by an anti-human IgG in an ELISA assay. The antibody titer (pg / ml) was determined for each heavy chain / light chain pair, and the titles with the different light chains of the rearranged germ line were compared with the titers obtained with the parental antibody molecule (i.e., the heavy chain paired with the analogous light chain), and the percentage of native titer was calculated (Table 2). VH: variable heavy chain gene. Vk: variable gene of the light chain k. ND: no expression detected under current experimental conditions.

Table 2

continuation

The results obtained from these experiments demonstrate that the somatically mutated heavy chains with high affinity of different gene families are capable of pairing with the Vk1-39Jk5 and Vk3-20Jk1 regions of the human germline rearranged and secreted from the cell as a normal antibody molecule. As shown in T abla 1, the antibody titer was increased by approximately 61% (42 of 69) of the heavy chains when paired with the rearranged human Vk1-39Jk5 light chain, and approximately 29% (20 of 69) of the heavy chains when paired with the rearranged human Vk3-20Jk1 light chain, compared to the analogous parental antibody light chain. For approximately 20% (14 of 69) of the heavy chains, both light chains of the rearranged human germ line conferred an increase in expression compared to the analogous light chain of the parental antibody. As shown in Table 2, the Vk1-39Jk5 region of the rearranged human germ line conferred an increase in the expression of various specific heavy chains for a range of different classes of antigens, compared to the analog light chain for antibodies. parental The antibody titer was more than doubled for approximately 35% (15/43) of the heavy chains, compared to the analogous light chain of the parental antibodies. For two heavy chains (315 and 316), the increase was more than ten times greater compared to the parental antibody. Within all the heavy chains that showed an increase in expression in relation to the parental antibody analog light chain, the heavy chains of family three (VH3) are overrepresented compared to other gene families of the variable region of heavy chain This demonstrates a favorable relationship of the human VH3 heavy chains to match the Vk1-39Jk5 and Vk3-20Jk1 light chains of the rearranged human germline.

Example 2

Generation of a locus of the rearranged human germline light chain

Various targeting vectors of the rearranged human germline line were made using VELOCIGENE® technology (see, for example, U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21 (6): 652-659), to modify clones 302g12 and 254m04 (Invitrogen) of mouse genomic Artificial Chromosome (BAC). Using these two BAC clones, genomic constructs were modified by genetic engineering to contain a single region of the rearranged human germline light chain and inserted into a locus of the endogenous light chain k that was previously modified to eliminate gene segments endogenous binding and variables k.

Construction of addressing vectors of the rearranged human germline light chain. Three different regions of the light chain of the rearranged human germ line were developed using standard molecular biology techniques recognized in the art. Human variable gene segments used to construct these three regions include a rearranged human Vk1-39Jk5 sequence, a rearranged human Vk3-20Jk1 sequence and a rearranged human VpreBJ 5 sequence.

A segment of DNA containing an exon 1 (encoding the leader peptide), and an intron 1 of the mouse Vk3-7 gene was performed by de novo DNA synthesis (Integrated DNA Technologies). Part of the 5 'untranslated region to the site of a naturally occurring Blpl restriction enzyme was included. The exons of the Vk1-39 and Vk3-20 genes were amplified by PCR from the human genomic BAC libraries. The direct primers showed an extension at the 5 'end that contained the splice acceptor site of intron 1 of the mouse Vk3-7 gene. The reverse primer used for the PCR of the human Vk1-39 sequence included an extension encoding the human Jk5, while the reverse primer used for the PCR of the human Vk3-20 sequence included an extension encoding the human Jk1. The human VpreBJA5 sequence was made by de novo DNA synthesis (Integrated DNA Technologies). A part of the human Jk-Ck intron that includes the cutting donor site and splicing, was amplified by PCR from plasmid pBS-296-HA18-PIScel. The direct primer included an extension that encodes part of either a Jk5, Jk1, or human JA5 sequence. The reverse primer included a Pl-Scel site, which was previously engineered in the intron.

The mouse exon / intron 1, the human variable light chain exons, and the human Jk-Ck intron fragments were linked together by overlapping PCR, digested with Blpl and Pl-Scel, and ligated into a pBS plasmid -296-HA18-PIScel, which contained the human Vk3-15 variable gene segment promoter. A hygromycin cassette with a LOX site inside plasmid pBS-296-HA18-PIScel was replaced by a hygromycin cassette with a FRT site flanked by Notl and Ascl sites. The Notl / Pl-Scel fragment of this plasmid was ligated into a modified mouse BAC 254m04, which contained part of the mouse Jk-Ck intron, the mouse Ck exon, and approximately 75 kb of genomic sequence downstream of the locus k of mouse, which provided a homology arm at the 3 'end for homologous recombination in mouse ES cells. The Notl / Ascl fragment of this BAC was then ligated into a modified mouse BAC 302g12, which contained a neomycin cassette with an FRT site and approximately 23 kb of water genomic sequence arrives from the endogenous locus for homologous recombination in ES cells of mouse.

Vk1-39Jk5 addressing vector of the rearranged human germ line (FlG. 1). Restriction enzyme sites were introduced at the 5 'and 3' ends of a genetically engineered light chain insert for cloning into a targeting vector: an Ascl site at the 5 'end and a Pl-Scel site at the 3 'end. Within the Ascl site of the 5 ′ end and the Pl-Scel site of the 3 ′ end the addressing construct from the 5 ′ to 3 ′ end included a 5 ′ homology arm containing a 5 ′ sequence with respect to the endogenous locus of the chain lightweight mouse k, obtained from mouse BAC clone 302g12, a neomycin resistance gene with an FRT site, a genomic sequence that includes the human Vk3-15 promoter, a leader sequence of the Vk3-7 variable gene segment of mouse, an intron sequence of the mouse Vk3-7 variable gene segment, an open reading frame of a Vk1-39Jk5 region of the rearranged human germ line, a genomic sequence containing a part of the human Jk-Ck intron, and a 3 'end homology arm containing a 3' sequence of the endogenous mouse Jk5 segment obtained from a mouse BAC 254m04 clone (Figure 1, center). The genes and / or sequences upstream of the endogenous locus of the mouse light chain k and downstream of the gene segment Jk plus 3 '(for example, endogenous enhancer 3'), were not modified by the targeting construct (see Figure 1 ). The sequence of the genetically engineered human Vk1-39Jk5 locus is shown in SEQ lD NO: 1.

Directed insertion of the Vk1-39Jk5 region of the rearranged human germ line into a BAC DNA was confirmed by polymerase chain reaction (PCR) using primers located in sequences within the light chain region of the human germ line. rearranged In summary, the sequence of introns 3 'with respect to the leader sequence of mouse Vk3-7 was confirmed with primers ULC-m1F (AGGTGAGGGT ACAGATAAGT GTTATGAG; SEQ lD NO: 2) and ULC-m1R (TGACAAATGC CCTAATTATA GTGATCA; SEQ l NO: 3). The open reading frame of the Vk1-39Jk5 region of the human germ line was confirmed with primers 1633-h2F (GGGCAAGTCA GAGCATTAGC A; SEQ lD NO: 4) and 1633-h2R (TGCAAACTGG ATGCAGCATA G; SEQ lD ^{NO: 5) . The neomycin cassette was confirmed with the neoF (GGTGGAGAGG CTATTCGGC; SEQ ID NO:} 6 ⁾ and neoR (GAACACGGCG GCATCAG; SEQ ID NO: 7) primers. The target BAC DNA was then used to electroporate mouse ES cells to create modified ES cells to generate chimeric mice that express a Vk1-39Jk5 region of the rearranged human germline.

Positive ES cell clones were confirmed by screening and karyotype studies of TAQMAN ™, using specific probes for the Vk1-39Jk5 region of the genetically modified light chain inserted into the ^{endogenous locus. Simply put, the neoP probe (TGGGCACAAC AGACAATCGG CTG; SEQ lD NO:} 8 ^{) that binds} within a neomycin marker gene, a ULC-m1P probe (CCATTATGAT GCTCCATGCC TCTCTGTTC; SEQ lD NO: 9) that binds within the intron sequence 3 'with respect to the leader sequence of mouse Vk3-7, and a 1633h2P probe (ATCAGCAGAA ACCAGGGAAA GCCCCT; SEQ lD NO: 10) that joins within the open reading frame of the rearranged Vk1-39Jk5 of the human germ line. Next, clones of positive ES cells were used to implant female mice to give rise to a litter of pups expressing the region of the Vk1-39Jk5 light chain of the germ line.

Alternatively, the ES cells that carry the Vk1-39Jk5 light chain region of the human germ line are transfected with a FLP-expressing construct to remove the neomycin cassette with an FRT site introduced by the targeting construct. Optionally, the neomycin cassette is removed by raising mice that express FLP recombinase (eg, US 6,774,279). Optionally, the neomycin cassette is maintained in the mice.

Vk3-20Jk1 redirected vector of the human germ line (FlG. 2). Similarly, a genetically modified light chain locus was performed that expresses a rearranged Vk3-20Jk1 region of the human germ line, using an addressing construct that included, from the 5 'to the 3' end, an arm of 5 'end homology containing a 5' sequence with respect to the endogenous locus of the mouse k light chain obtained from a mouse BAC 302g12 clone, a neomycin resistance gene with an FRT site, a genomic sequence which included the human Vk3-15 promoter, a leader sequence of the mouse Vk3-7 variable gene segment, an intron sequence of the mouse Vk3-7 variable gene segment, a reading frame open of a rearranged Vk3-20Jk1 region of the human germ line, a genomic sequence that contained a part of the human Jk-Ck intron, and a homology arm at the 3 'end that contained a 3' sequence of the mouse Jk5 gene segment obtained from a mouse BAC 254m04 clone (Figure 2, center). The sequence of the genetically engineered human Vk3-20Jk1 locus is shown in SEQ ID NO: 11.

Directed insertion of the rearranged Vk3-20Jk1 region of the human germline into BAC DNA was confirmed by polymerase chain reaction (PCR), using primers located in sequences within the region of the rearranged Vk3-20Jk1 light chain region. of the human germ line. Simply put, the 3 'intron sequence with respect to the leader sequence of mouse Vk3-7 was confirmed with primers ULCmlF (SEQ ID NO: 2) and ULC-m1R (SEQ ID NO: 3). The open reading frame of the rearranged Vk3-20Jk1 region of the human germ line was confirmed with primers 1635-h2F (TCCAGGCACC CTGTCTTTG; SEQ ID NO: 12) and 1635-h2R (AAGTAGCTGC TGCTAACACT CTGACT; SEQ ID NO: 13) . The neomycin cassette was confirmed with the ^{neoF (SEQ ID NO:} 6 ^{) and neoR (SEQ ID NO: 7) primers . Targeted BAC DNA was then used to electroporate} ES mouse cells to ES cells created modified to generate chimeric mice expressing the rearranged Vk3-20Jk1 light chain of the human germ line.

Positive ES cell clones were confirmed by screening and karyotype study of TAQMAN ™ using specific probes for the Vk3-20Jk1 light chain region genetically engineered inserted into the ^{endogenous locus of the light chain k. In summary, a neoP probe (SEQ ID NO:} 8 ^{) that binds within the} neomycin marker ^gene , a ULC-m1P probe (SEQ ID NO: 9) that binds within the leader sequence of the mouse Vk3-7, and a 1635h2P probe (AAAGAGCCAC CCTCTCCTGC Ag GG; SEQ ID NO: 14) that joins within the open reading frame of human Vk3-20Jk1. Next, clones of positive ES cells were used to implant female mice. There was a litter of young that express the region of the light chain Vk3-20Jk1 of the human germ line.

Alternatively, ES cells that carry a region of the Vk3-20Jk1 light chain of the human germ line can be transfected with a construct that expresses FLP, to remove the neomycin cassette with an FRT site introduced by the targeting construct. Optionally, the neomycin cassette can be removed by raising mice that express FLP recombinase (eg, US 6,774,279). Optionally, the neomycin cassette is maintained in the mice.

VpreBJA5 addressing vector rearranged from the human germ line (FIG. 3). Similarly, a genetically modified light chain locus was performed that expresses a rearranged VpreBJA 5 region of the human germ line, using a targeting construct that included, from end 5 'to end 3', a homology arm at the 5 'end containing a 5' sequence with respect to the endogenous locus of the mouse light chain k obtained from a mouse BAC 302g12 clone, a neomycin resistance gene with an FRT site, a genomic sequence that it included the human Vk3-15 promoter, a leader sequence of the mouse Vk3-7 variable gene segment, an intron sequence of the mouse Vk3-7 variable gene segment, an open reading frame of a rearranged VpreBJA5 region of the human germ line , a genomic sequence containing a part of the human Jk-Ck intron, and a 3 'end homology arm containing a 3' sequence of the endogenous mouse Jk5 gene segment obtained from a mouse BAC 254m04 clone (Figure 3, center). The sequence of the genetically engineered human VpreBJ 5 locus is shown in SEQ ID NO: 15.

Directed insertion of the rearranged VpreBJ 5 region of the human germ line into BAC DNA was confirmed by polymerase chain reaction (PCR), using primers located in sequences within the region of the rearranged VpreBJA5 light chain region. human germinal Simply put, the 3 'intron sequence with respect to the leader sequence of mouse Vk3-7 was confirmed with primers ULCmlF (SEQ ID NO: 2) and ULC-m1 R (SEQ ID NO: 3). The open reading frame of the rearranged VpreBJA5 region of the human germ line was confirmed with primers 1616-h1F (TGTCCTCGGC Cc Tt GGA; SEQ ID NO: 16) and 1616-h1R (CCGATGTCAT GGTCGTTCCT; SEQ ID NO: 17). The neomycin cassette was confirmed with the neoF ^{(SEQ ID NO:} 6 ^{) and neoR (SEQ ID NO: 7)} primers ^{. Targeted BAC DNA was then used to electroporate} ES mouse cells to ES cells created modified to generate chimeric mice expressing the rearranged VpreBJA5 light chain of the human germ line.

Positive ES cell clones were confirmed by screening and karyotype study of TAQMAN ™ using specific probes for the genetically modified region of the VpreBJA5 light chain inserted into the ^{endogenous locus of the light chain k. In summary, a neoP probe (SEQ ID NO:} 8 ^{) that binds within the} neomycin marker ^gene , a ULC-m1P probe (SEQ ID NO: 9) that binds within the leader sequence of the mouse Vk3-7, and a 1616h1P probe (ACAATCCGCC TCACCTGCAC CCT; SEQ ID NO: 18) that joins within the open reading frame of human VpreBJA5. Next, clones of positive ES cells were used to implant female mice to give rise to a litter of offspring expressing a region of the germline light chain. Alternatively, ES cells that carry a region of the rearranged VpreBJ 5 light chain of the human germ line can be transfected with a construct that expresses FLP, to remove the neomycin cassette with an FRT site introduced by the targeting construct. Optionally, the neomycin cassette can be removed by raising mice that express FLP recombinase (eg, US 6,774,279). Optionally, the neomycin cassette is maintained in the mice.

Example 3

Generation of mice expressing a single rearranged human light chain

The target ES cells described above were used as donor ES cells and were introduced into a ^mouse embryo ^{in a state of} 8 ^{cells by the VELOCIMOUSE® method (see, for example, US Pat. No. 7,294,754} and Poueymirou et al. . (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses, Nature Biotech. 25 (1): 91-99.). VELOCIMICE® mice that independently carry a region of the Vk1-39Jk5 light chain of the human germ line, a region of the Vk3-20Jk1 light chain or a region of the VpreBJ 5 light chain are identified by genotyping using a specific assay modification of allele (Valenzuela et al., supra) that detects the presence of the only region of the light chain of the rearranged human germline.

The offspring are genotyped and a heterozygous or homozygous offspring are selected for the single region of the rearranged human germline light chain, to characterize the expression of the rearranged human germline light chain region.

Flow cytometry . The expression of the rearranged human light chain region in the normal antibody repertoire of common light chain mice was validated by analysis of the expression of immunoglobulin k and A in splenocytes and in peripheral blood of common light chain mice. Cell suspensions of cultured spleens and peripheral blood were made from wild-type mice (n = 5), heterozygous common light chain mice Vk1-39Jk5 (n = 3), homozygous common light chain Vk1-39Jk5 (n = 3), Vk3-20Jk1 common light chain heterozygotes (n = 2), and Vk3-20Jk1 common light chain homozygotes (n = 2), using standard methods and stained with CD19 +, IgA + and IgK + using fluorescently labeled antibodies (BD Pharmigen) .

^{In summary, 1x10} 6 ^{were incubated with anti-mouse CD16 / CD32 (clone 2.4G2, BD Pharmigen) on ice for 10 minutes,} followed by labeling with the following antibody cocktail for 30 minutes on ice: anti-mouse CD19 conjugated to APC (clone 1D3, BD Pharmigen), anti-mouse CD3 conjugated to PerCP-Cy5.5 (clone 17A2, BioLegend), IgK anti mouse conjugated to FITC (clone 187.1, BD Pharmigen), anti-mouse IgA conjugated to PE (clone RML-42, BioLegend). Following staining, the cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on a flow cytometer and analyzed with FlowJo. Limitation of subpopulations of interest (Gating): total B lymphocytes (CD19 + CD3-), IgK + B lymphocytes (IgK + IgA-CD19 + CD3-), IgA + B lymphocytes (IgK-IgA + CDl9 + CD3-). Data collected from blood samples and splenocytes showed similar results. Table 3 shows the percentage of CD19 + B lymphocytes positive for peripheral blood of a representative mouse of each group, which are IgA +, IgK +, or IgA + IgK +. The percentage of CD19 + B lymphocytes in peripheral blood of wild type or wild type mice (WT) and homozygous for the common light chain Vk1-39Jk5 or Vk3-20Jk1 are shown in FIG. Four.

Table 3

Common light chain expression. The expression of each common light chain (Vk1-39Jk5 and Vk3-20Jk1) was analyzed in heterozygous and homozygous mice using a quantitative PCR assay (for example, from TAQMAN ™).

In summary, CD19 + B lymphocytes were purified from homozygous wild-type mouse spleens for a replacement of the heavy chain locus and the variable region of the mouse light chain k, with the loci of the variable region of the mouse Light chain k and corresponding human heavy chain (Hk), in addition to homozygous and heterozygous mice for each region of the rearranged human light chain (Vk1-39Jk5 or Vk3-20Jk1) using mouse CD19 microbeads (Miltenyi Biotec) according to Manufacturer's specifications. Total CD19 + B lymphocyte RNA was purified using a RNeasy Mini Kit (from Qiagen) according to the manufacturer's specifications and genomic RNA was removed using RNase-free DNase column treatment (Qiagen). 200 ng mRNA were reverse transcribed into cDNA using the First Strand cDNA synthesis kit (Invitrogen) and the resulting cDNA was amplified with the Taqman Universal PCR Master Mix (Applied Biosystems). All reactions were developed using the ABI 7900 sequence detection system (Applied Biosystems), using MGB Taqman MGB primers and probes that span (1) the Vk-Jk link for both common light chains, (2) the Vk gene alone (ie Vk1-39 and Vk3-20), and (3) the mouse Ck region. Table 4 shows the sequences of the primers and probes used for this assay. Relative expression was normalized to the expression of the mouse Ck region. The results are shown in FIG. 5A, 5B and 5C.

____________________ ________________ Table 4_______________________ _____________

| Region | Description of the Primer / Probe (5'-3 ') | I KNOW THAT. ID Nos: |

Common light chain antigen specific antibodies. Common light chain mice that carried either a common light chain Vk1-39Jk5 or Vk3-20Jk1 were immunized in the endogenous locus of the mouse k light chain, with p-galactosidase and the antibody titer was measured.

In summary, p-galactosidase (Sigma) was emulsified in titermax adjuvant (Sigma), following the manufacturer's instructions. Wild-type mice (n = 7), homozygous common light chain Vk1-39Jk5 (n = 2) and homozygous common light chain Vk3-20Jk1 (n = 5) were immunized by subcutaneous injection with 100 µg pgalactosidase / Titermax Mice were reinforced twice by subcutaneous injection, at 3-week intervals, with 50 jg of p-galactosidase / Titermax. After the second booster, blood was collected from anesthetized mice using retro-orbital bleeding in serum separator tubes (BD Biosciences) according to the manufacturer's instructions. To measure IgM or IgG anti-p-galactosidase antibodies, ELISA plates (Nunc) were coated with 1 jg / ml p-galactosidase overnight at 4 ° C. The excess antigen was washed before blocking it with PBS with 1% BSA for one hour at room temperature. Serial dilutions were added to the plates and incubated for one hour at room temperature before washing. The plates were then incubated with anti-IgM (Southern Biotech) or anti-IgG (Southern Biotech) conjugated with HRP for one hour at room temperature. Following another wash, the plates were developed with TMB substrate (BD Biosciences). The reactions were stopped with 1 N sulfuric acid and OD450 readings were taken using a Victor X5 Plate Reader (Perkin Elmer). The data was analyzed with GraphPad Prism and the signal was calculated as the serum dilution that is twice ^{the background. The results are shown in FIG.} 6 ^{A and} 6 ^B.

As shown in this example, the ratio of B k / A lymphocytes in both the splenic and peripheral compartments of mice of the common light chain Vk1-39Jk5 and Vk3-20Jk1 demonstrated a pattern close to the wild type (Table 3 and FIG. 4). The mice of the common light chain VpreBJA5, however, demonstrated fewer ^peripheral B lymphocytes ^{, of which approximately} 1-2 ^{% express the region of the human light chain modified by} genetic engineering (data not shown). The expression levels of the regions of the rearranged human light chain Vk1-39Jk5 and Vk3-20Jk1 of the endogenous locus of the light chain k were high compared to an endogenous locus of the light chain k containing a complete replacement of the gene segments Mouse Vk and Jk with human Vk and Jk gene segments (FIG. 5A, 5B and 5C). Expression levels of the VpreBJ 5 rearranged human light chain region demonstrated similar elevated expression of the endogenous light chain locus k in both heterozygous and homozygous mice (data not shown). This demonstrates that in direct competition with the endogenous loci of the mouse light chain A, k, or both, a single human rearranged VL / JL sequence can produce a better level of expression than the wild type of the endogenous light chain locus k and give rise to a normal frequency of blood and splenic B lymphocytes. In addition, the presence of a locus of the light chain k genetically engineered with a human Vk1-39Jk5 or Vk3-20Jk1 sequence was well tolerated by mice and appears to function in the form of wild type representing a substantial part of the repertoire of the ^{light chain in the humoral component of the immune response (FIG} 6 ^{A and} 6 ^B).

Example 4

Breeding of mice expressing a single light chain of the rearranged human germ line

This example describes several different strains of a genetically modified mouse that can be engendered in any of the common light chain mice described in the present patent to create multiple genetically modified mouse strains that house multiple genetically modified immunoglobulin locus.

Knockout (KO) of the IgA. To optimize the use of the genetically modified light chain locus, mice bearing regions of the rearranged human germline light chain are generated in another mouse that contains a deletion in the endogenous light chain locus A. in this way, the progeny obtained will express, as its only light chain, the region of the light chain of the rearranged human germ line, as described in Example 2. The breeding is carried out by standard techniques recognized in the art and, alternatively, through a commercial breeder (for example, The Jackson Laboratory). Mouse strains that carry a genetically engineered light chain locus and a deletion of the endogenous light chain A locus are screened to detect the presence of the single region of the light chain and the absence of light chains A endogenous mouse.

Humanized endogenous heavy chain locus. Mice carrying a genetically engineered human germline heavy chain locus are engendered with mice that contain a locus replacement of the endogenous mouse heavy chain variable gene with the heavy chain variable gene locus human (see US 6,596,541; the VELOCIMMUNE® mouse, Regeneron Pharmaceuticals, Inc.). The VELOCIMMUNE® mouse consists of a genome comprising variable regions of the human heavy chain operatively linked to the loci of the endogenous mouse constant region, such that the mouse produces antibodies comprising a variable region of the human heavy chain and a constant region of the mouse heavy chain in response to antigenic stimulation. The DNA encoding the variable regions of the heavy chains of the antibodies is isolated and operably linked to the DNA encoding the constant regions of the human heavy chain. The DNA is then expressed in a cell capable of expressing the completely human chain of the antibody.

Mice bearing a replacement of the endogenous mouse Vh locus with the human Ch locus and a single Vl region of the human germline rearranged in the endogenous k light chain locus are obtained. Inverse chimeric antibodies are obtained containing somatically mutated heavy chains (human Vh and mouse Ch) with a single human light chain (human Vl and mouse Cl Cl) after immunization with an antigen of interest. The Vh and Vl nucleotide sequences of the B lymphocytes expressing antibodies are identified and fully human antibodies are made by fusion of the Vh and Vl nucleotide sequences with the human Ch and Cl nucleotide sequences in a suitable expression system.

Example 5

Generation of antibodies from mice expressing human heavy chains and a region of the rearranged human germline light chain

After breeding mice containing the genetically engineered human light chain region to obtain various strains containing modifications and deletions of other endogenous Ig locuses (as described in Example 4), the selected mice can be immunized with a antigen of interest

Generally, a VELOCIMMUNE® mouse containing one of the regions of the single light chain of the rearranged human germ line is exposed to an antigen, and lymphatic cells (such as B lymphocytes) are recovered from the animals' serum. The lymphatic cells are fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies containing variable regions of the heavy chain and light chains of the rearranged human germ line that are specific to the antigen used for immunization. The DNA encoding the variable regions of the heavy chains and the light chain is isolated and linked to desirable isotypic constant regions of the heavy chain and the light chain. Due to the presence of endogenous mouse sequences and any additional cis-acting elements present in the endogenous locus, the single light chain of each antibody can be somatically mutated. This adds additional diversity to the specific repertoire of the antigen comprising a single light chain and various heavy chain sequences. The sequences of the resulting cloned antibody are subsequently expressed in a cell, such as a CHO cell. Alternatively, the DNA encoding the antigen specific chimeric antibodies or the domains of the light and heavy chains are directly identified from antigen specific lymphocytes.

Initially, high affinity chimeric antibodies with a human variable region and a mouse constant region are isolated. As described above, antibodies are characterized and selected to exhibit desirable characteristics, including affinity, selectivity, epitope, etc. Mouse constant regions are replaced with a desired human constant region to generate the fully human antibody that contains a somatically mutated human heavy chain and a single light chain obtained from a region of the human germline light chain of the invention. . The appropriate human constant regions they include, for example, wild-type or modified IgG1 or IgG4.

Different cohorts of VELOCIMMUNE® mice were immunized containing a replacement of the endogenous mouse heavy chain locus with human VH, DH, and JH gene segments, and a replacement of the endogenous mouse light chain k locus or with the region of the human light chain Vk1-39Jk5 of the genetically engineered germline, or with the region of the human light chain Vk3-20Jk1 of the genetically engineered germline (described above), with a surface receptor protein cell (E antigen). The E antigen is administered directly in the plant of the paw of the mice with six consecutive injections every 3-4 days. Two or three Antigen E microorganisms are mixed with 10 | jg of CpG oligonucleotide (Cat # tlrl-modn oligonucleotide - ODN1826; InVivogen, San Diego, CA) and 25 jg of Adju-Phos (aluminum phosphate gel adjuvant, Cat # H-71639-250; Brenntag Biosector, Frederikssund, Denmark) prior to injection. A total of six injections are provided prior to the final withdrawal of the antigen, which is administered 3-5 days ^{prior to sacrifice. Blood extractions are collected after the 4th and} 6th ^{injection and the} antibody's immune ^{response is monitored} by a standard antigen specific immunoassay.

When a desired immune response is achieved, splenocytes are harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. Hybridoma cell lines are screened and selected to identify cell lines that produce antibodies to the common light chain specific for the E antigen. Using this technique, various specific antibodies against the common light chain antigen are obtained (i.e. antibodies that possess variable domains of the human heavy chain, the same variable domain of the human light chain, and constant mouse domains).

Alternatively, anti-Antigen E antibodies of the common light chain are isolated from B-positive lymphocytes in antigens without fusion with myeloma cells, as described in U.S. Pat. 2007 / 0280945A1, specifically incorporated herein by reference in its entirety. Using this method, various fully human common light chain anti-Antigen E antibodies were obtained (i.e., antibodies that possess variable domains of the human heavy chain, either a region of the genetically engineered human Vk1-39Jk5 light chain or a region of the human-engineered Vk3-20Jk1 light chain and human constant domains)

The biological properties of the examples of common light chain anti-Antigen E antibodies generated in accordance with the methods of this Example are described in detail in the sections set forth below.

Example 6

Use of a heavy chain gene segment in the antigen-specific common light chain antibodies

In order to analyze the structure of the common anti-human E-antigen light chain antibodies produced, nucleic acids encoding variable regions of heavy chain antibodies were cloned and sequenced. From the nucleic acid sequences and amino acid sequences of the predicted antibodies, the use of genes for the heavy chain variable region (HCVR) of the selected common light chain antibodies obtained from VELOCIMMUNE® mice immunized containing either the ^{genetically engineered human Vk1-39Jk5} light chain region or the genetically engineered human Vk3-20Jk1 light chain region ^{. The results are shown in Tables 5 and} 6 ^{, which} demonstrate that mice according to the invention generate antigen-specific light chain antibodies from a variety of human heavy chain gene segments, due to a variety of rearrangements, when using either a mouse that expresses a light chain from only a human Vk1-39 or a light chain obtained from Vk3-20-human. The human VH gene segments of families 2, 3, 4, and 5 are rearranged with a variety of human DH segments and JH segments to produce antigen specific antibodies.

Table 5

^{T l} 6

continuation

Example 7

Determination of the blocking of the ability of antigen-specific common light chain antibodies by assay with LUMINEX ™

Ninety-eight human common light chain antibodies generated against the E antigen were tested for their ability to block the binding of the natural ligand of Antigen E (Ligand Y) to the E antigen in a pearl test.

^{The extracellular domain (ECD) of the E antigen was conjugated with two myc epitope tags and a} 6 ^X histidine ^tag (E-mmH Antigen), and coupled with amine to carboxylated microspheres at a concentration of 20 pg / ml in buffer. MONTH. The mixture was incubated for two hours at room temperature, followed by deactivation of beads with 1 M Tris pH 8.0, followed by washing in PBS with 0.05% Tween-20 (v / v). The beads were then blocked with PBS (Irvine Scientific, Santa Ana, CA) containing 2% (w / v) BSA (Sigma-Aldrich Corp., St. Louis, MO). In a 96-well filter plate, supernatants containing antibodies of the common light chain specific to Antigen E were diluted in a 1:15 ratio in buffer solution. A negative control was prepared containing a mock supernatant with the same media components as for the antibody supernatant. Pearls labeled with antigen E were added to the supernatants and incubated overnight at 4 ° C. A protein with a biotinylated Ligand Y was added at a final concentration of 0.06 nM and incubated for two hours at room temperature. The detection of biotinylated Ligand Y bound to beads labeled with E-myc-myc-6 ^His Antigen ^{was determined with R-Phycoerythrin conjugated to Streptavidin (Moss Inc, Pasadena, MD), followed by measurement} in a LUMINEX ™ cytometry-based analyzer flow. The mean background fluorescence intensity (MFl) of a sample without Ligand Y was subtracted from all samples. The blocking percentage was calculated by dividing the MFI subtracted from the bottom of each sample by the adjusted negative control value, multiplying by 100 ^{and subtracting the resulting value from} 100 ^.

In a similar experiment, the same common light chain antibodies generated against Antigen E were tested to determine their ability to block binding of Antigen E to beads labeled with Ligand Y.

In summary, Ligand Y was coupled with amine to carboxylated microspheres in a concentration of 20 pg / ml diluted in an MES buffer. The mixture was incubated for two hours at room temperature followed by ^{deactivation of beads with 1 M Tris pH} 8 ^{, and then washed in PBS with 0.05% Tween-20 (v / v). The beads} were then blocked with PBS (Irvine Scientific, Santa Ana, CA) containing 2% (w / v) BSA (Sigma-Aldrich Corp., St. Louis, MO). In a 96-well filter plate, supernatants containing antibodies of the common light chain specific to Antigen E were diluted in a 1:15 ratio in buffer solution. A negative control was prepared containing a mock supernatant with the same media components as for the antibody supernatant. Pearls labeled with antigen E were added to the supernatants and incubated overnight at 4 ° C. A biotinylated EmmH Antigen was added to a final concentration of 0.42 nM and incubated for two hours at room temperature. Pearls labeled with Ligand Y were then added to the antibody / antigen mixture and incubated for two hours at room temperature. The detection of biotinylated E-mmH Antigen bound to Ligand Y was determined with R-Phycoerythrin conjugated to Streptavidin (Moss Inc, Pasadena, MD), followed by measurement in a LUMINEX ™ analyzer based on flow cytometry. The mean background fluorescence intensity (MFI) of a sample without Antigen E was subtracted from all samples. The blocking percentage was calculated by dividing the MFI subtracted from the bottom of each sample by the ^adjusted negative control value ^{, multiplying by} 100 ^{and subtracting the resulting value from} 100 ^.

^{Tables 7 and} 8 ^{show the percentage of blockade for all 98 anti-E antibodies E of the} common ^{light chain} tested in both tests with LUMINEX ™. ND: not determined under current experimental conditions.

Table 7

continuation

continuation

^{T l} 8

In the first experiment with LUMINEX ™ described above, 80 common light chain antibodies containing the genetically engineered Vk1-39Jk5 light chain were tested to determine their ability to block ligand Y binding to beads labeled with the E. Antigen Of these 80 antibodies of the ^{common light chain,} 68 ^{demonstrated a blockade of> 50%, while 12 demonstrated a blockade of <50%} (6 ^{a blockade of 25-50% and} 6 ^{a blockade of <25% ). For the 18 antibodies of the common light chain containing the genetically engineered Vk3-20Jk1 light chain, 12 demonstrated a block of> 50%, while} 6 demonstrated a block of <50% (3 a block of 25-50% and 3 a block of <25%) of the binding of Ligand Y to the beads marked with Antigen E.

In the second experiment with LUMINEX ™ described above, the same 80 common light chain antibodies that contain the genetically modified Vk1-39Jk5 light chain were tested to determine their ability to block binding of Antigen E to labeled beads with Ligand Y. Of these 80 antibodies of the common light chain, 36 demonstrated a blockade of> 50%, while 44 demonstrated a blockade of <50% (27 a blockade of 25-50% and 17 a blockade of < 25%) For the 18 antibodies of the ^{common light} chain ^{containing the genetically engineered Vk3-20Jk1 light chain,} 1 ^{demonstrated a block of} > 50%, while 17 demonstrated a block of <50% (5 a block of 25-50% and 12 a block of <25%) of the binding of Antigen E to the beads marked with Ligand Y.

^{The data in Tables 7 and} 8 ^{establish that the rearrangements described in Tables 5 and} 6 ^{generated antibodies} Anti-Antigen E specific to the common light chain that blocked the binding of Ligand Y to its analogous receptor Antigen E, with varying degrees of efficacy, which is compatible with the anti-Antigen E antibodies of the ^{common light} chain ^{of the Tables 5 and} 6 ^{comprising antibodies with epitope specificity with overlap and without} overlap with respect to Antigen E.

Example 8

Determination of the ability to block antigen specific common light chain antibodies by ELISA

The human common light chain antibodies generated against Antigen E were tested for their ability to block binding of Antigen E to a surface coated with a Ligand Y in an ELISA.

Ligand Y was coated on 96-well plates in a concentration of 2 pg / ml diluted in PBS and incubated overnight, followed by a four-fold wash in PBS with 0.05% Tween-20. The plate was then blocked with PBS (Irvine Scientific, Santa Ana, CA) containing 0.5% (w / v) BSA (Sigma-Aldrich Corp., St. Louis, MO) for one hour at room temperature. In a separate plate, supernatants containing ^{anti-Antigen E} antibodies ^{of the common light chain} were diluted ^{in a ratio of} 1:10 ^{in buffer solution. A} mock ^supernatant with the same antibody components was used as a negative control. E-mmH Antigen (described above) was added to a final concentration of 0.150 nM and incubated at room temperature. The antibody / E-mmH antigen mixture was then added to the plate containing Ligand Y, and incubated for one hour at room temperature. Detection of E-mmH Antigen bound to Ligand Y was determined with horseradish peroxidase (HRP) conjugated to an anti-Penta-His antibody (Qiagen, Valencia, CA) and developed by colorimetric response using tetramethylbenzidine (TMB) substrate (BD) Biosciences, San Jose, CA) neutralized by sulfuric acid. The absorbance in OD450 was read for 0.1 sec. The absorbance of a sample without Antigen E was subtracted from all samples. The blocking percentage was calculated by dividing the MFI subtracted from the bottom of each ^{sample by the value of the adjusted negative control, multiplying by} 100 ^{and subtracting the resulting value from} 100 ^.

Tables 9 and 10 show the percentage of blockage during all 98 light chain antibodies tested in the ELISA. ND: not determined under current experimental conditions.

Table 9

continuation

Table 10

As described in this Example, of the 80 antibodies of the common light chain containing the genetically modified Vk1-39Jk5 light chain, were tested to determine their ability to block the binding of Antigen E to a surface coated with the Ligand Y, 22 demonstrated a blockade of> 50%, while 58 demonstrated a blockade of <50% (20 a blockade of 25-50% and 38 a blockade of <25%). For the 18 antibodies of the common light chain containing the genetically engineered Vk3-20Jk1 light chain, one demonstrated a block of> 50%, while 17 demonstrated a block of <50% (5 a block of 25-50% and 12 a block of <25%) of the binding of Antigen E to a surface coated with Ligand Y.

These results are also compatible with the set of common light chain antibodies specific for Antigen E comprising antibodies with epitope specificity with overlap or without overlap with respect to Antigen E.

Example 9

Determination of affinity with BIACORE ™ of common light chain antibodies specific for the antigen

Balance dissociation constants (Kd) were determined for antibody supernatants selected by SPR (Surface Plasmon Resonance) using a BIACORE ™ T100 instrument (GE Healthcare). All data were obtained using Hb S-EP (10 mM Hepes, 150 mM NaCl, 0.3 mM and Dt A, 0.05% P20 surfactant, pH 7.4) both as a migration buffer and a loading buffer, at 25 ° C. The antibodies were captured from samples of crude supernatant on the surface of a CM5 sensor chip previously derived with antibodies with high-density anti-human Fc antibodies using standard amine coupling chemistry. During the capture stage, the supernatants were injected through the anti-human Fc surface at a flow rate of 3 pl / min, for a total of 3 minutes. The capture stage was followed by an injection of either migration buffer or analyte, at a concentration of 100 nM for 2 minutes, at a flow rate of 35 pl / min. ^{The antigen dissociation from the captured antibody was monitored for} 6 ^{minutes. The} captured ^antibody was removed by a brief injection of 10 mM glycine, pH 1.5. All sensograms were referenced twice by subtracting the sensograms of the buffer injections from the analyte sensograms, thereby eliminating artifacts caused by dissociation of the antibody from the capture surface. ^Binding data ^{for each antibody was fitted to a model in a} 1: 1 ^{ratio with mass transport using the} BIAcore T100 Evaluation software v2.1. The results are shown in Tables 11 and 12.

____________ Table 11____________

Common Light Chain Antibodies

Vk1-39Jk5

Vk1-39Jk5

00nM of 100 nM Antigen E Antibody Antigen E Antibody

Kd nM T1 / 2 min Kd nM T1 / 2 min

n e n e n geno

Antibody Antigen E_____ Antibody ____________________ __________ Kd (nM) T1 / 2 (min) ___________ Kd (nM) T1 / 2 (min) ^{2968 5.50} 8 2973 5.35 39 ^{2968G 305 2973G} 11.0 ^{44 2969 34.9 2974 256} 0 ^{2969G 181 2974G 138} 0

12,3

²⁹⁷⁵

^38,0

2 ____________________________ (continuación)______________2970G

12.3

²⁹⁷⁵

^38.0

two ____________________________ (continuation)______________

Common Light Chain Antibodies

_____________________________ Vk3-20Jk1 ________________

100 nM of 100 n

Antibody Antigen E_____ Antibody ______

Kd (nM) T1 / 2 (min) ___________ Kd (n

^{2971G 32.8 134}

²⁹⁷² 6.02 ^6.7

^74,6

⁶⁵⁶

^2972G

^74.6

⁶⁵⁶

The binding affinities of the common light chain antibodies comprising the rearrangements shown ^{in Tables 5 and} 6 ^{vary, where practically all show a Kd in the nanomolar range. The affinity data} are compatible with the common light chain antibodies that are the result of the combinatorial association of ^{rearranged variable domains described in Tables 5 and} 6 ^{that are high affinity, clonally selected, and} somatically mutated. Coupled to the data shown previously, the common light chain antibodies ^{described in Tables 5 and} 6 ^{comprise a collection of various high affinity antibodies that show the} specificity for one or more epitopes in Antigen E.

Example 10

The determination of the binding specificities of the common light chain antibodies specific for the antigen by assay with LUMINEX ™

Anti-E antibodies of the common light chain were tested for their ability to bind ECD of Antigen E and ECD variants of Antigen E, including the cynomolgus monkey ortholog (Mf Antigen E), which differs from human protein in approximately 10% of its amino acid residues; an E Antigen deletion mutant lacks the last 10 amino acids of the C-terminal end of the ECD (E-ACT Antigen); and two mutants that contain an alanine substitution at suspected locations of interaction with Ligand Y (E-Alal Antigen and E-Ala2 Antigen). Antigen E proteins were produced in CHO cells and each contained a C-terminal myc-myc-His tag.

For binding studies, an ECD protein of Antigen E or variant protein (described above) ^of 1 ^{ml of culture medium} was captured ^{by incubation for} 2 h at ^{room temperature with} 1 ^x 1 ^O 6 covalently coated ^{beads of} microspheres (LUMINEX ™) with an anti-myc monoclonal antibody (MAb 9ElO, hybridoma cell line CRL-1729 ™; ATCC, Manassas, VA). The beads were then washed with PBS before use. Supernatants containing anti-E antibodies of the common light chain were diluted in a 1: 4 ratio in a buffer solution and added to 96-well filter plates. A mock supernatant without any antibody was used as a negative control. The beads containing the captured Antigen E proteins were then added to the antibody samples (3OOO beads per well) and incubated overnight at 4 ° C. The next day, the sample beads were washed, and the bound common light chain antibody was detected with an anti-human antibody conjugated to R-phycoerythrin. The fluorescence intensity of the beads (approximately 1OO beads counted for each antibody sample that binds to each Antigen E protein), was measured with a LUMINEX ™ flow cytometry analyzer, and the average fluorescence intensity was recorded ( MFI) for at least 1OO beads counted per pearl / antibody interaction. The results are shown in Tables 13 and 14.

Table 13

continuation

Table 14

Claims (20)

1. A genetically modified mouse comprising a B lymphocyte that expresses a variable domain (Vl) of the human light chain obtained from a rearranged human Vk1-39 / Jk sequence that is present in the mouse germ line, where the mouse lacks an endogenous non-rearranged immunoglobulin Vk gene segment and an endogenous non-rearranged immunoglobulin Jk gene segment; and wherein the human Vl domain (or domains) is associated with a variable domain (Vh) of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region selected from 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6-13 / 4. 2. The mouse of claim 1, wherein: (a) the human Vk1-39 / Jk sequence is operably linked to a sequence of the mouse Ck region; (b) a sequence encoding the human Vh domain is operably linked to a sequence encoding a sequence of the constant region (Ch) of the mouse heavy chain selected from a Ch1, a hinge, a CH2, a CH3, or a combination thereof; or (c) the human VH domain is expressed from an endogenous immunoglobulin heavy chain locus. 3. The mouse of claim 1, wherein all or substantially all of the mouse Vk and Jk gene segments are replaced by the rearranged human Vk1-39 / Jk sequence. 4. The mouse of claim 1, wherein the rearranged human Vk1-39 / Jk sequence is operably linked to a sequence of the constant region of the immunoglobulin light chain selected from a mouse, rat or human sequence. 5. A genetically modified mouse comprising a B lymphocyte that expresses a variable domain (Vl) of the human light chain obtained from a rearranged human Vk3-20 / Jk sequence that is present in the mouse germ line, where the mouse lacks a Vk endogenous gene segment of non-rearranged immunoglobulin and a Jk endogenous gene segment of non-rearranged immunoglobulin; and wherein the human Vl domain (or domains) is associated with a variable domain (Vh) of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region selected from 3-30 / 3-3 / 3, 3-33 / 1-7 / 4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4. 6 ^{. The mouse of claim 5, wherein:} (a) the human Vk3-20 / Jk sequence is operably linked to a sequence of the mouse Ck region; (b) a sequence encoding the human Vh domain is operably linked to a sequence encoding ^{a sequence of the mouse region Ch selected from a Ch1, a hinge, a Ch} 2 ^{, a Ch3, or a} combination thereof; or (c) the human VH domain is expressed from an endogenous immunoglobulin heavy chain locus. 7. The mouse of claim 5, wherein all or substantially all of the mouse Vk and Jk gene segments are replaced by the rearranged human Vk3-20 / Jk sequence. 8 ^{. The mouse of claim 5, wherein the rearranged human Vk3-20 / Jk sequence is operably linked to} a sequence of the constant region of the immunoglobulin light chain selected from a mouse, rat or human sequence. 9. The mouse of any of the preceding claims, wherein the mouse comprises a non-functional lambda (A) immunoglobulin light chain locus. 10. Use of the mouse of any one of claims 1-4 in the manufacture of an antibody comprising a variable domain (Vh) of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region selected from 2- 5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3- 30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1- 69 / 6-13 / 4 associated with a variable region of the human light chain obtained from a rearranged human Vk1-39 / Jk sequence. 11. The use of claim 10, wherein the antibody is a bispecific antibody. 12. Use of the mouse of any one of claims 5-8 in the manufacture of an antibody comprising a variable domain (VH) of the human heavy chain obtained from a rearranged human VH / DH / JH region selected from 3- 30 / 3-3 / 3, 3-33 / 1-7 / 4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4 associated with a variable region of the human light chain obtained from of a rearranged human Vk3-20 / Jk sequence. 13. The use of claim 12, wherein the antibody is a bispecific antibody. 14. The use of any of claims 11 and 13, wherein the production is by a method that It comprises the steps of expressing the variable domains of the human heavy chain in a single cell. 15. The use of any one of claims 10-14, wherein the antibody heavy chains are completely human. 16. The use of any one of claims 10 or 11, wherein the heavy chains comprising the variable domains of the heavy chain are paired with a light chain comprising the human Vl domain obtained from a sequence Vk1-39 / Jk human rearranged. 17. The use of any one of claims 12 or 13, wherein the heavy chains comprising the variable domains of the human heavy chain are paired with a light chain comprising the human VL domain obtained from a Vk3-20 sequence / Jk human rearranged. 18. The use of any one of claims 10-17, wherein the variable domain (or domains) of the human heavy chain is selected by immunizing the mouse with an antigen of interest, determining the sequences of the variable domains of the heavy chain human expressed by the mouse, and expressing the sequences in the cell. 19. A method of producing an antibody to an antigen of interest comprising a human heavy chain variable domain obtained from a rearranged human Vh / Dh / Jh region selected from 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6-13 / 4 associated with a region human light chain variable obtained from a rearranged human Vk1-39 / Jk sequence, the method comprising: (a) immunizing the mouse of any one of claims 1-4 with an antigen of interest; (b) obtain from the mouse a variable domain of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region selected from 2-5 / 3-22 / 1, 3-13 / 6-6 / 5, 3-23 / 2-8 / 4, 3-23 / 6-6 / 4, 3-23 / 7-27 / 4, 3-30 / 1-1 / 4, 3-30 / 3-3 / 4, 3-30 / 5-5 / 2, 3-30 / 7-27 / 6, 1-69 / 6-6 / 5 or 1-69 / 6-13 / 4; Y (c) employing the sequence of the immunoglobulin variable region obtained in (b) associated with said human light chain variable region in an antibody that specifically binds to the antigen of interest. 20. A method of producing an antibody for an antigen of interest comprising a human heavy chain variable domain obtained from a rearranged human Vh / Dh / Jh region selected from 3-30 / 3-3 / 3, 3 -33 / 1-7 / 4, 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4 associated with a variable region of the human light chain obtained from a human Vk3-20 / Jk sequence reordered, understanding the method: (a) immunizing the mouse of any one of claims 5-8 with an antigen of interest; (b) obtain from the mouse a variable domain of the human heavy chain obtained from a rearranged human Vh / Dh / Jh region selected from 3-30 / 3-3 / 3, 3-33 / 1-7 / 4 , 3-33 / 2-15 / 4 or 3-53 / 1-1 / 4; Y (c) employing the sequence of the immunoglobulin variable region obtained in (b) associated with said human light chain variable region in an antibody that specifically binds to the antigen of interest.